Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 2021/102107
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THERAPEUTIC ADENO-ASSOCIATED VIRUS COMPRISING LIVER-SPECIFIC
PROMOTERS FOR TREATING POMPE DISEASE AND LYSOSOMAL DISORDERS
SEQUENCE LISTING
[0001] This invention claims benefit under 35 U.S.C.
119(e) of U.S. Provisional Application
62/937,556 filed on November 19, 2019, and U.S. Provisional Application
62/937,583 filed on
November 19, 2019, and U.S. Provisional Application 63/023,570 filed on May
12, 2020, the contents
of each are incorporated herein in their entirety by reference.
SEQUENCE LISTING
100021 The instant application contains a Sequence Listing which has been
submitted electronically
in ASCII format, and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on
November 17, 2020, is named 046192-096600WOPT SL.txt and is 840,179 bytes in
size.
FIELD OF THE INVENTION
MOW] The present invention relates to adeno-associated virus (AAV) particles,
virions and vectors
for targeted translocation of lysosomal enzymes, such as, e.g., an alpha-
glucosidase (GALA)
polypeptide, and method of use for the treatment of lysosomal storage diseases
and disorders, such as,
e.g., Pompe disease.
BACKGROUND
[0004] More than forty lysosomal storage diseases (LSDs) are caused, directly
or indirectly, by the
absence of one or more lysosomal enzymes in the lysosome Enzyme replacement
therapy for LSDs is
being actively pursued. Therapy generally requires that LSD proteins be taken
up and delivered to the
lysosomes of a variety of cell types in an M6P-dependent fashion. One possible
approach involves
purifying an LSD protein and modifying it to incorporate a carbohydrate moiety
with M6P. This
modified material may be taken up by the cells more efficiently than
umnodified LSD proteins due to
interaction with M6P receptors on the cell surface.
[0005] As an alternative or adjunct to enzyme therapy, the feasibility of gene
therapy approaches to
treat GSD-II have been investigated (Amalfitano, A., et al., (1999) Proc.
Natl. Acad. Sc!. USA
96:8861-8866, Ding, E., et al. (2002)MoL Ther. 5:436-446, Fraites, T. J., et
al., (2002)MoI. Ther.
5:571-578, Tsujino, S., et al. (1998) Than. Gene Ther. 9:1609-1616).
[0006] However, viral or AAV delivery of genes, in particular lysosomal
proteins and enzymes for
treatment of lysosomal storage diseases has challenges. Normally, mammalian
lysosomal enzymes are
synthesized in the cytosol and traverse the ER where they are glycosylated
with N-linked, high
marmose type carbohydrate. In the Golgi, the high marmose carbohydrate is
modified on lysosomal
proteins by the addition of mannose-6-phosphate (M6P) which targets these
proteins to the lysosome.
The M6P-modified proteins are delivered to the lysosome via interaction with
either of two M6P
receptors. However, recombinantly produced proteins used in enzyme replacement
therapy often lack
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the addition of the M6P which is required for targeting them to the lysosomes,
therefore, often
requiring high doses of recombinantly produced enzymes to be administered to a
patient and/or
frequent infusions.
[0007] Acid alpha-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes the
alpha 1-4 linkage
in maltose and other linear oligosaccharides, including the outer branches of
glycogen, thereby
breaking down excess glycogen in the lysosome (Hirschhorn et al. (2001) in The
Metabolic and
Molecular Basis of Inherited Disease, Scriver, et al., eds. (2001), McGraw-
Hill: New York, p. 3389-
3420). Like other mammalian lysosomal enzymes, GAA is synthesized in the
cytosol and traverses
the ER where it is glycosylated with N-linked, high mannose type carbohydrate.
In the Golgi, the high
mannose carbohydrate is modified on lysosomal proteins by the addition of
mannose-6-phosphate
(M6P) which targets these proteins to the lysosome. The M6P-modified proteins
are delivered to the
lysosome via interaction with either of two M6P receptors. The most favorable
form of modification
is when two M6Ps are added to a high mannose carbohydrate.
[0008] Insufficient GAA activity in the lysosome results in Pompe disease, a
disease also known as
acid maltase deficiency (AMD), glycogen storage disease type II (GSDII),
glycogenosis type II, or
GAA deficiency. The diminished enzymatic activity occurs due to a variety of
missense and nonsense
mutations in the gene encoding GAA. Consequently, glycogen accumulates in the
lysosomes of all
cells in patients with Pompe disease. In particular, glycogen accumulation is
most pronounced in
lysosomes of cardiac and skeletal muscle, liver, and other tissues.
Accumulated glycogen ultimately
impairs muscle fimction. In the most severe form of Pompe disease, death
occurs before two years of
age due to cardio-respiratory failure.
[0009] There is a need for an effective treatment of Pompe disease. Enzyme
replacement
therapeutics for Pompe require a recombinant GAA protein to be administered
and taken up by
muscle and liver cells in the subject where it is subsequently transported to
the lysosomes in those
cells in a M6P-dependent fashion. However, while enzyme therapy has
demonstrated reasonable
efficacy for severe infantile GSD II, the benefit of GAA enzyme therapy is
limited by the need for
frequent infusions as well as the subject developing inhibitor or neutralizing
antibodies against
recombinant hGAA protein (Amalfitano, A., et al. (2001) Genet In Med 3:132-
138).
[0010] Gene therapy has the potential to not only cure genetic disorders, but
to also facilitate the
long-term non-invasive treatment of acquired and degenerative disease using a
virus. One gene
therapy vector is adeno-associated virus (AAV). AAV itself is a non-pathogenic-
dependent
parvovims that needs helper viruses for efficient replication. AAV has been
utilized as a virus vector
for gene therapy because of its safety and simplicity. AAV has a broad host
and cell type tropism
capable of transducing both dividing and non-dividing cells.
[0011] However, AAV delivery of the GAA polypeptide has some challenges with
respect to
achieving sufficient expression in the liver and/or delivery to lysosomes with
patients reporting to
experience glycaemia.
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In particular, in human subjects, the administration of rAAV vectors encoding
GAA polypeptide have
resulted in a number of patients experiencing hypoglycemia or becoming
hyperglycemic due to non-
specific update in cells (see, e.g., Byrne et al, A study on the safety and
efficacy of Reveglucosidease
alfa in patients with late-onset Pompe disease; Orphanet J. of Rare diseases;
2017; 12: 144).
[0012] Accordingly, there is a need in the art for improved methods of
producing lysosomal
polypeptides such as GAA in vitro and in vivo, for example, to treat lysosomal
polypeptide
deficiencies, including modifications of GAA. Moreover, there is a need for
improved secretion from
the liver as well as improved targeting of GAA to the lysosomes to help reduce
any side effects from
overexpression of the GAA polypeptide, and reducing the risk of hypoglycemia.
Further, there is a
need for methods that result in systemic delivery of GAA and other lysosomal
polypeptides to
affected tissues and organs. In particular, there remains a need for more
efficient methods for
administering GAA protein to subjects and targeting GAA protein to patient
lysosomes, while
reducing any potential side effects.
SUMMARY OF THE INVENTION
[0013] The technology described herein relates generally to gene therapy
constructs, methods and
composition, for the treatment lysosomal storage diseases and disorders, such
as, for example but not
limited to, Pompe Disease. More particularly, the technology relates to adeno-
associated (AAV)
virions configured for delivering a lysosomal enzyme, e.g., a GAA polypeptide
to a subject, and more
particularly for delivering a lysosomal enzyme, e.g., a GAA polypeptide to the
liver of a subject
where it is targeted to the lysosomes and secreted from the liver cells.
100141 In particular, described herein are targeted viral vectors, e.g., using
rAAV vectors as an
exemplary example, that comprise a nucleotide sequence containing inverted
terminal repeats (ITRs),
a promoter, a heterologous gene, a poly-A tail and potentially other regulator
elements for use to treat
a lysosomal storage disease, such as those listed in Table 5A or Table 6A
herein, wherein the
heterologous gene is a lysosomal enzyme, such as, e.g., GAA, and wherein the
vector, e.g., rAAV can
be administered to a patient in a therapeutically effective dose that is
delivered to the appropriate
tissue and/ or organ for expression of the heterologous lysosomal enzyme gene
and treatment of the
disease, e.g., Pompe disease.
[0015] Aspects of the present invention teach certain benefits in construction
and use which give
rise to the exemplary advantages described below.
[0016] Accordingly, in particular embodiments described herein are rAAV
vectors that comprises a
nucleotide sequence containing inverted terminal repeats (ITRs) and located
between the ITRs, a liver
specific promoter (LSP), a heterologous nucleic acid sequence that encodes the
acid alpha-glucosidase
(GAA) protein, a poly-A tail and potentially other regulator elements for use
to treat Pompe Disease,
and wherein the rAAV expressing GAA protein can be administered to a patient
in a therapeutically
effective dose that is delivered to the appropriate tissue and/or organ for
expression of the
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heterologous gene encoding the GAA protein for the treatment of a subject with
Pompe disease.
100171 More specifically, the AAV virion or genome comprises a LSP selected
from any promoter
listed in Table 4 herein, or a functional variant or functional fragment
thereof, or any LSP selected
from SEQ ID NO: 86, 91-96, or 146-150 or a functional variant or functional
fragment thereof, that
enables the lysosomal protein, e.g., GAA protein to be preferentially
expressed in the liver. In some
embodiments, the liver-specific promoter, while preferentially expresses the
hGAA protein in the
liver, can also express the hGAA to some extent in another tissue of interest,
e.g., the muscle, or CNS,
or muscle and CNS tissues. In some embodiments, the expressed lysosomal
enzyme, e.g., GAA
protein can be configured as GAA-fusion protein with a targeting sequence,
such as a IGF2 targeting
peptide as disclosed herein that targets the GAA protein to lysosomes, and/or
fused with a signal
peptide (SP), the GAA protein is expressed by the rAAV genome in the liver,
where it is secreted and
taken up by lysosomes of mammalian cells, in particular muscle cells.
100181 In some embodiments of the compositions and methods described herein,
the rAAV vector
disclosed herein comprises, in its genome: 5' and 3' AAV inverted terminal
repeats (ITR) sequences,
and located between the 5' and 3' ITRs, a liver specific promoter (LSP)
operatively linked to a
heterologous nucleic acid sequence encoding an alpha-glucosidase (GAA)
polypeptide, wherein the
liver-specific promoter (LSP) comprises a nucleic acid sequence selected from
any promoter listed
from SEQ ID NOS: 86 (CRM 0412), SEQ ID NO: 91 (5P0412) or SEQ ID NO: 92
(5P0422), SEQ ID
NOS: 93 (SP0239), SEQ ID NO: 94 (5P0265, also referred to SP131_Al), SEQ ID
NO: 95 (SP0240)
or SEQ ID NO: 96 (5P0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147
(5P0239-UTR),
SEQ ID NO: 148 (5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150
(SP0131-Al-
UTR) or a functional fragment or variant thereof, or any LSP selected from SEQ
ID NO: 270-341 or
342-430, or a functional fragment or variant thereof. In some embodiments, the
GAA polypeptide is
not fused to either a IGF2 tnrgeting sequence, or signal sequence. In some
embodiments, the GAA
polypeptide is fused to a signal sequence as disclosed herein, and/or a IGF2
targeting sequence as
disclosed herein.
100191 In some embodiments of the compositions and methods described herein,
the rAAV vector
disclosed herein comprises, in its genome: 5' and 3' AAV inverted terminal
repeats (ITR) sequences,
and located between the 5' and 3' ITRs, a liver specific promoter (LSP)
operatively linked to a
heterologous nucleic acid sequence encoding a fusion polypeptide comprising
(i) a secretory signal
peptide, and/or an IGF2 targeting peptide; and (ii) an alpha-glucosidase (GAA)
polypeptide, wherein
the liver-specific promoter (LSP) is selected from any promoter listed in
Table 4 herein, or a
functional variant or functional fragment thereof, or any LSP selected from
SEQ ID NO: 86, 91-96, or
146-150 or a functional variant or functional fragment thereof.
100201 In some embodiments, the rAAV vector disclosed herein comprises, in its
genome: 5' and
3' AAV inverted terminal repeats (ITR) sequences, and located between the 5'
and 3' ITRs, a
heterologous nucleic acid sequence encoding a fusion polypeptide comprising
(i) a secretory signal
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peptide (also referred to as a leader peptide), and (ii) an alpha-glucosidase
(GAA) polypeptide,
wherein the heterologous nucleic acid is operatively linked to a liver-
specific promoter (LSP) selected
from any promoter listed in Table 4 herein, or a functional variant or
functional fragment thereof, or
any LSP selected from SEQ ID NO: 86, 91-96, or 146-150, or a functional
variant or functional
fragment thereof or any LSP selected from Table 4 herein, or a functional
variant or functional
fragment thereof. Exemplary leader sequences include, but are not limited to
the innate GAA leader
sequence, AAT sequence, IL2(1-3), IL2 leader sequence (IL2 wt), a modified IL2
leader sequence
(IL2 mut), fibronectin (FN1) signal sequence, or IgG leader sequence or
functional variants thereof, as
disclosed herein. In some embodiments, the AAV vector comprises a Kozak
sequence located
between the LSP and the leader sequence.
100211 In some embodiments, the rAAV vector disclosed herein comprises, in its
genome: 5' and
3' AAV inverted terminal repeats (ITR) sequences, and located between the 5'
and 3' ITRs, a
heterologous nucleic acid sequence encoding a fusion polypeptide comprising
(i) an IGF2 targeting
peptide, and (ii) an alpha-glucosidase (GAA) polypeptide, wherein the
heterologous nucleic acid is
operatively linked to a liver-specific promoter (LSP) selected from any
promoter listed in Table 4
herein, or a functional variant or functional fragment thereof, or any LSP
selected from SEQ ID NO:
86, 91-96, or 146-150 or a functional variant or functional fragment thereof.
100221 In a further embodiments, the rAAV vector disclosed herein comprises,
in its genome: 5'
and 3' AAV inverted terminal repeats (ITR) sequences, and located between the
5' and 3' ITRs, a
heterologous nucleic acid sequence encoding an alpha-glucosidase ((fAA)
polypeptide (i.e., where the
GAA polypeptide not fused to a heterologous signal peptide (or a leader
sequence), or not fused to an
IGF2 targeting sequence as described herein), wherein the heterologous nucleic
acid is operatively
linked to a liver-specific promoter (LSP) selected from any promoter listed in
Table 4 herein, or a
functional variant or functional fragment thereof, or any LSP selected from
SEQ ID NO: 86, 91-96,01
146-150 or a functional variant or functional fragment thereof.
100231 In some embodiments, the rAAV vector comprises a liver specific capsid,
e.g., a liver
specific capsid selected from XL32 and XL32.1, as disclosed in W02019/241324,
which is
incorporated herein in its entirety by reference. In some embodiments, the
rAAV vector is a
AAVXL32 or AAVXL32,1 as disclosed in W02019/241324, which is incorporated
herein in its
entirety by reference, or a AAV8 vector, or a haploid AAV vector comprising at
least one AAV8
capsid protein (e.g., at least one of VP1, VP2, or VP3 is from the AAV8
serotype), and in some
embodiments, the AAV vector is a haploid AAV vector comprising at least two
AAV8 capsid
proteins). In some embodiments, the AAV vector comprises a capsid disclosed in
W02019241324A1,
or International Patent application PCT/US2019/036676, which are incorporated
herein in their
entirety by reference. In some embodiments, the AAV vector comprises a capsid
which is encoded by
a nucleic acid AAV capsid coding sequence that is at least 90% identical to a
nucleotide sequence of
any one of SEQ ID NOs: 1-3 as disclosed in W02019241324A1; or (b) a nucleotide
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encoding any one of SEQ ID NOS:4-6 as disclosed in W02019241324A1. In some
embodiments, an
AAV capsid comprises an amino acid sequence at least 90% identical to any one
of SEQ ID NOS:4-6
as disclosed in W02019241324A1, along with AAV particles comprising an AAV
vector genorne and
the AAV capsid of the invention.
In some embodiments, the rAAV vector comprises capsid proteins such that the
AAV vector
transduces liver cells, and in some embodiments the rAAV vector comprises the
rAAV vector
comprises capsid proteins such that the AAV vector transduces muscle and liver
cells.
[0024] An exemplary LSP encompassed for use in the methods and compositions is
SP0412 (SEQ
ID NO: 91) or a functional variant thereof. In alternative embodiments, a LSP
can be selected from
any of SEQ ID NOS: 86 (CRM 0412), SEQ ID NO: 91 (5P0412) or SEQ ID NO: 92
(5P0422), SEQ
ID NOs: 93 (5P0239), SEQ ID NO: 94 (5P0265, also referred to SP13 l_Al), SEQ
ID NO: 95
(SP0240) or SEQ ID NO: 96 (5P0246), or SEQ ID NO: 146 (SP0265-UTR), SEQ ID NO:
147
(SP0239-UTR), SEQ ID NO: 148 (SP0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ
ID NO:
150 (SP0131-A1-UTR), or functional fragments or variants thereof.
[0025] In some embodiments of the compositions and methods described herein,
the secretory
signal peptide is selected from any of AAT signal peptide, a fibronectin
signal peptide (FM!), a GAA
signal peptide, innate GAA leader sequence, AAT sequence, IL2(1-3), IL2 leader
sequence (IL2 wt),
a modified IL2 leader sequence (IL2 mut), or IgG leader sequence or functional
variants thereof
having secretory signal activity.
[0026] In some embodiments of the compositions and methods described herein,
the alpha-
glucosidase (GAA) polypeptide is linked to the IGF2 targeting peptide at the N-
terminal end of a
GAA polypeptide. In some embodiments, the IGF2 targeting peptide is linked to
the N-terminal at
amino acid 70 of human acid alpha-glucosidase ((MA) polypeptide (SEQ ID NO:
10) (i.e., linked to
the N-terminal of residues 70-952 of human acid alpha-glucosidase (GAA)
polypeptide), or a GAA
polypeptide at least 85% sequence identity to amino acids 70-952 of SEQ ID NO:
10. In alternative
embodiments, the IGF2 targeting peptide is linked to the N-terminal at amino
acid 40 of human acid
alpha-glucosidase (GAA) polypeptide (SEQ ID NO: 10) (i.e., linked to the N-
terminal of residues 40-
952 of human acid alpha-glucosidase (GAA) polypeptide) ), or a GAA polypeptide
at least 85%
sequence identity to amino acids 40-952 of SEQ ID NO: 10. In some embodiments
of the
compositions and methods described herein, the GAA polypeptide is encoded by
the wild-type GAA
nucleic acid sequence (e.g., SEQ ID NO: 11 or SEQ ID NO: 72), or can be a
codon optimized GAA
nucleic acid sequence, e.g., for any one of increasing expression in vivo,
reducing CpG islands and/or
reducing innate immune response in a subject. Exemplary codon optimized GAA
nucleic acid
sequences include, but are not limited to SEQ ID NO; 73, SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID
NO: 76 and SEQ ID NO: 182.
[0027] In some embodiments of the methods and compositions disclosed herein,
the recombinant
AAV vector comprises a liver-specific promoter (LSP), for example but not
limited to, a liver specific
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promoter is selected from any in Table 4 herein or functional variants
thereof, or functional variants
thereof. Exemplary LSP encompassed for use in the methods and compositions
include SP0412 and
functional variants thereof. In alternative embodiments, a LSP can comprise a
nucleic acid sequence
selected from any of SP0422, SP0131A1, SP0239, SP0240 or SP0246, or a
functional variant thereof
as disclosed herein. For Example, the liver specific promoter can comprise a
nucleic acid sequence
selected from any of SEQ ID NOS: 86 (CRM 0412), SEQ ID NO: 91 (5P0412) or SEQ
ID NO: 92
(SP0422), or a functional variant or functional fragment thereof. In
alternative embodiments, the liver
specific promoter can comprise a nucleic acid sequence selected from any of
SEQ ID NOs: 93
(5P0239), SEQ ID NO: 94 (5P0265 also called SP13 l_Al), SEQ ID NO: 95 (5P0240)
or SEQ ID
NO: 96 (SP0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR),
SEQ ID
NO: 148 (5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR), or SEQ ID NO: 150 (SP0131-A
1-UTR).
In some embodiments of the compositions and methods disclosed herein, a liver-
specific promoter,
includes a liver-specific cis-regulatory element (CRE), a synthetic liver-
specific cis-regulatory module
(CRM) or a synthetic liver-specific promoter comprising a promoter sequence
selected from any of
SEQ ID NOs: 270-341 (minimal LSP, which can include a CRM) or SEQ ID NO: 342-
430(exemplary
synthetic LSP), or a functional fragment or functional variant thereof, as
previously disclosed in
Tables 4A or 4B of provisional application 62,937,556, which is encompassed in
its entirety by
reference herein. These liver-specific promoter elements can include minimal
liver-specific promoters
(see, e.g., SEQ ID NO: 86, 270-341 or liver-specific proximal promoters (see,
e.g., SEQ ID Nos: 91-
96, 146-150 and 342-430). For Example, SEQ ID NOs: 86 (CRM 0412), SEQ ID NO:
91 (SP0412) or
SEQ ID NO: 92 (5P0422), or a functional variant or functional fragment thereof
100281 In some embodiments of the methods and compositions disclosed herein,
the recombinant
AAV vector comprises a liver-specific promoter (LSP), for example but not
limited to, a liver specific
promoter is selected from any of SEQ ID NOs: 86, 91-96, 146-150, 370-430 or a
functional variant or
functional fragment thereof.
100291 For example, a functional variant or a functional fragment of a liver-
specific promoter
disclosed in Table 4 herein, or any LSP selected from SEQ ID NO: 86, 91-96, or
146-150, or 370430
or a functional variant or functional fragment thereof has at least about 75%
sequence identity to, or at
least about 80% sequence identity to, at least about 90% sequence identity to,
at least about 95%
sequence identity to, at least about 98% sequence identity to the original
unmodified reference
sequence, and also at least 35% of the promoter activity, or at least about
45% of the promoter
activity, or at least about 50% of the promoter activity, or at least about
60% of the promoter activity,
or at least about 75% of the promoter activity, or at least about 80% of the
promoter activity, or at
least about 85% of the promoter activity, or at least about 90% of the
promoter activity, or at least
about 95% of the promoter activity of the corresponding unmodified promoter
sequence.
100301 For example, a functional variant or a functional fragment of SEQ ID
NO: 92 (5P0422) or
SEQ ID NO: 91 (SP0412) has at least about 75% sequence identity to SEQ ID NO:
92 or SEQ ID
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NO: 91, or at least about 80% sequence identity to SEQ ID NO: 92 or SEQ ID NO:
91, at least about
90% sequence identity to SEQ ID NO: 92 or SEQ ID NO: 91, at least about 95%
sequence identity to
SEQ ID NO: 92 or SEQ ID NO: 91, at least about 98% sequence identity to SEQ ID
NO: 92 or SEQ
ID NO: 91, or the original unmodified sequence, and also at least 35% of the
promoter activity, or at
least about 45% of the promoter activity, or at least about 50% of the
promoter activity, or at least
about 60% of the promoter activity, or at least about 75% of the promoter
activity, or at least about
80% of the promoter activity, or at least about 85% of the promoter activity,
or at least about 90% of
the promoter activity, or at least about 95% of the promoter activity of the
corresponding unmodified
promoter sequence of SEQ ID NO: 92 or SEQ ID NO: 91, respectively.
100311 A functional fragment is a portion of the promoter that has at least
35%, or at least about
45%, or at least about 50%, or at least about 75%, or at least about 80%, or
at least about 85%, or at
least about 90% of the untrunkated promoter In some embodiments, a functional
fragment comprises
a contiguous portion of the unmodified promoter sequence. While TTR (SEQ ID
NO: 431) is
disclosed in the Examples herein as an exemplary LSP, one of ordinary skill in
the art can replace the
TTR promoter (SEQ ID NO: 431) with any one or more of the liver-specific
promoter listed in Table
4 herein, for example, a nucleic acid sequence comprising at least SEQ ID NO:
92 (SP0422) or SEQ
ID NO: 91 (SP0412) or a functional variant or fragment of SEQ ID NO: 92
(SP0422) or SEQ ID NO:
91 (SP0412), or a nucleic acid sequence comprising any of SEQ ID NOs: 93
(5P0239), SEQ ID NO:
94 (SP131_Al), SEQ ID NO: 95 (5P0240), SEQ ID NO: 96 (SP0246), or SEQ ID NO:
146 (5P0265-
UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID NO: 148 (5P0240-UTR), SEQ ID NO: 149
(5P0246-UTR) or SEQ ID NO: 150 (SP0131-A1-UTR), or any sequence selected from
SEQ ID NO:
270-341 or 342-430, or a functional variant or functional fragment thereof. In
some embodiments, the
LSP, while preferentially expresses the hGAA protein in the liver, can also
express the hGAA to some
extent in another tissue of interest, e.g., the muscle, or CNS, or muscle and
CNS tissues.
100321 In some embodiments of the methods and compositions disclosed herein, a
recombinant
AAV vector comprises a heterologous nucleic acid sequence that encodes a wild-
type GAA
polypeptide (wtGAA) or a modified GAA polypeptide, as disclosed herein, where
one or more amino
acids of the GAA polypeptide is modified, e.g., H199R, R223H, H201L
modifications. In some
embodiments of the methods and compositions disclosed herein, a recombinant
AAV vector
comprises a heterologous nucleic acid sequence encoding the GAA polypeptide
that is the human
GAA gene or a human codon optimized GAA gene (coGAA) or a modified GAA nucleic
acid
sequence that is codon optimized that encodes a modified GAA polypeptide
comprising one or more
of the modifications selected from: H199R, R223H, H201L. In all aspects of the
methods and
compositions as disclosed herein, a nucleic acid sequence encoding the GAA
polypeptide is codon
optimized for any one or more of enhanced expression in vivo, to reduce CpG
islands, or to reduce
the innate immune response. In all aspects of the methods and compositions as
disclosed herein, a
nucleic acid sequence encoding the GAA polypeptide is codon optimized to
reduce CpG islands and
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to reduce the innate immune response. In some embodiments, the nucleic acid
sequence encoding the
wild type GAA polypeptide comprises modifications as disclosed in SEQ ID NO:
182, and described
herein.
100331 Another aspect of the technology herein relates to a pharmaceutical
composition comprising
any of the recombinant AAV vector compositions disclosed herein, and a
pharmaceutically acceptable
carrier.
100341 Another aspect of the technology herein relates to a composition
comprising a nucleic acid
sequence comprising in the following order a 5' Inc a liver specific promoter
(LSP) operatively
linked to a nucleic acid sequence comprising a nucleic acid encoding a
modified GAA polypeptide
comprising one or more of the modifications selected from: H199R, R223H,
H201L, and a 3' ITR. In
one aspect the nucleic acid sequence optionally further comprises a nucleic
acid sequence encoding a
leader sequence (or signal sequence) located between the LSP and the nucleic
acid encoding the GAA
polypeptide, where the leader sequence is selected from any of: the innate GAA
leader sequence,
AAT sequence, IL2(1-3), IL2 leader sequence (IL2 wt), a modified IL2 leader
sequence (IL2 mut),
fibronectin (FN1), or IgG leader sequence or functional variants thereof, as
disclosed herein. In some
embodiments, the nucleic acid sequence optionally further comprises a kozak
sequence located
between the LSP and the leader sequence. In some embodiments, the nucleic acid
sequence optionally
further comprises am IGF2 targeting peptide located between the leader
sequence and the nucleic acid
encoding the GAA polypeptide. In some embodiments, the nucleic acid sequence
optionally further
comprises a 3' UTR located 3' of the nucleic acid encoding the GAA polypeptide
and the polyA
sequence. In some embodiments, the nucleic acid sequence optionally further
comprises an intron
sequence 3' of the LSP and 5' of the nucleic acid encoding the GAA
polypeptide, preferably between
the LSP and the kozak sequence. Exemplary constructs for the rAAV vector or
rAAV genome are
shown in FIGS. 5A-5G.
100351 Another aspect of the technology herein relates to a composition
comprising a nucleic acid
sequence comprising a 5' ITR, a liver specific promoter (LSP) operatively
linked to a nucleic acid
sequence encoding a modified GAA polypeptide comprising one or more of the
modifications
selected from: H199R, R223H, H201L, a polyA sequence and a 3' Mt sequence,
where the poly A
sequence can be a full length or truncated polyA signal sequence. Another
aspect of the technology
herein relates to a composition comprising a nucleic acid sequence comprising
a 5' ITR, a liver
specific promoter (LSP) operatively linked to a nucleic acid sequence encoding
a modified GAA
polypeptide comprising one or more of the modifications selected from: H199R,
R223H, H201 L, a
full-length polyA sequence, a terminal repeat sequence and a 3' ITR sequence,
where the nucleic acid
lacks a AAV P5 promoter sequence.
100361 Another aspect of the technology herein relates to a composition
comprising a nucleic acid
sequence comprising: a liver specific promoter (LSP) operatively linked to a
nucleic acid sequence
comprising, in the following order: (a) a nucleic acid encoding a secretory
signal peptide, (b) a nucleic
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acid encoding a IGF2 targeting peptide, and (c) a nucleic acid encoding a GAA
polypeptide.
[0037] Another aspect of the technology herein relates to a composition
comprising a nucleic acid
sequence for a recombinant adenovims associated (rAAV) vector genome, the
nucleic acid sequence
comprising: (a) a 5' and a 3' AAV inverted terminal repeats (ITR) nucleic acid
sequences, and (b)
located between the 5' and 3' ITR sequence, a heterologous nucleic acid
sequence encoding a
polypeptide comprising a secretory signal peptide and an alpha-glucosidase
((1AA) polypeptide,
wherein the heterologous nucleic acid is operatively linked to a liver-
specific promoter as described
above. An exemplary liver-specific promoter is 5P0412 or 5P0422 or a
functional variant thereof. In
some embodiments, a liver-specific promoter for use in the methods and
compositions as disclosed
herein includes a liver-specific cis-regulatory element (CRE), a synthetic
liver-specific cis-regulatory
module (CRM) or a synthetic liver-specific promoter as disclosed in Table 4
herein.
100381 In some embodiments of the methods and compositions disclosed herein,
the nucleic acid
sequence comprises a heterologous nucleic acid sequence encoding a GAA
polypeptide, where the
nucleic acid sequence is a human GAA gene or a human codon optimized GAA gene
(coGAA) or a
modified GAA nucleic acid sequence. In some embodiments of the methods and
compositions
disclosed herein, the nucleic acid sequence comprises a heterologous nucleic
acid sequence that is a
codon optimized (coGAA) GAA gene, for any one or more of enhanced expression
in vivo, to reduce
CpG islands or to reduce the innate immune response. In some embodiments of
the methods and
compositions disclosed herein, the nucleic acid sequence comprises a
heterologous nucleic acid
sequence that is a codon optimized (coGAA) GAA gene to reduce CpG islands and
to reduce the
innate immune response.
[0039] In some embodiments of the methods and compositions disclosed herein,
the nucleic acid
sequence comprises a heterologous nucleic acid sequence encoding a GAA
polypeptide selected from
any of SEQ ID NO: 11 (full length hGAA), SEQ ID NO: 55 (Dwight cDNA), SEQ ID
NO: 56
(hGAA A1-66) or SEQ ID NO: 182 (modGAA, H199R, R223H), or a nucleic acid
sequence encoding
a GAA polypeptide having the amino acid sequence of SEQ ID NO: 170 (modGAA;
H199R, R223H),
SEQ ID NO: 171 (modGAA; H199R, R223H, H201L), or a nucleic acid sequence
encoding a GAA
polypeptide that is at least about 75%, or 80%, or 85%, or 90%, or 95%, or
98%, or 99% sequence
identity to any of SEQ ID NOs: 11, 55, 56 or 182.
[0040] In some embodiments of the methods and compositions disclosed herein,
the nucleic acid
sequence comprises a heterologous nucleic acid sequence encoding the GAA
polypeptide, where the
nucleic acid encoding the GAA polypeptide is selected from any of SEQ ID NO:
74 (codon optimized
1), SEQ ID NO: 75 (codon optimized 2), and SEQ ID NO: 76 (codon optimized 3),
or SEQ ID NO:
182 (modGAA, H199R, R223H), or a nucleic acid sequence at least about 75%, or
80%, or 85%, or
90%, or 95%, or 98%, or 99% sequence identity to any of SEQ ID NOs: 74, 75, 76
or 182.
[0041] Another aspect of the technology herein relates to use of the rAAV and
nucleic acid
compositions disclosed herein in a method to treat a disease. In particular,
one aspect of the
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technology herein relates to use of the rAAV vector compositions and nucleic
acid compositions
disclosed herein, in a method to treat a subject with a glycogen storage
disease type II (GSD II,
Pompe Disease, Acid Maltase Deficiency) or having a deficiency in alpha-
glucosidase (GAA)
polypeptide, the method comprising administering any of the recombinant AAV
vector, or the rAAV
genome or the nucleic acid sequence disclosed herein to the subject. In some
embodiments of the
methods disclosed herein, the expressed GAA polypeptide is secreted from the
subject's liver and
there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle
tissue, diaphragm
muscle tissue or a combination thereof, wherein uptake of the secreted GAA
results in a reduction in
lysosomal glycogen stores in the tissue(s). In some embodiments in the
disclosed methods, the
recombinant AAV vector, or the rAAV genome or the nucleic acid sequence is
administered to the
subject by any suitable administration method, for example, but not limited
to, an administration
method selected from any of: intramuscular, sub-cutaneous, intraspinal,
intracistemal, intrathecal,
intravenous administration. In some embodiments, the pharmaceutical
composition disclosed herein
can be used in the methods disclosed herein.
[0042]
Another aspect of the technology
herein relates to a cell comprising any one or more of a
rAAV composition, a rAAV genome composition, or a nucleic acid composition as
disclosed herein.
In some embodiments, the cell is a human cell, or a non-human cell mammalian
cell, or an insect cell.
[0043] Another aspect of the technology herein relates to host animal
comprising any one or more
of a rAAV composition, a rAAV genome composition, or a nucleic acid
composition as disclosed
herein. In some embodiments, the host animal is a mammal, a non-human mammal
or a human.
[0044] Another aspect of the technology herein relates to host animal
comprising at least one cell
that comprises any one or more of a rAAV composition, a rAAV genome
composition, or a nucleic
acid composition as disclosed herein. In some embodiments, the host animal
comprising such a
modified cell is a mammal, a non-human mammal or a human.
[0045] In some embodiments, disclosed herein is a pharmaceutical formulation
comprising an
rAAV vectors, nucleic acid encoding a rAAV genome as disclosed herein, and a
pharmaceutically
acceptable carrier.
[0046] Aspects of the present invention teach certain benefits in construction
and use which give
rise to the exemplary advantages described below. Other features and
advantages of aspects of the
present invention will become apparent from the following more detailed
description, taken in
conjunction with the accompanying drawings, which illustrate, by way of
example, the principles of
aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] This application file contains at least one drawing executed in color.
Copies of this patent
application publication with color drawings will be provided by the Office
upon request and payment
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of the necessary fee. The accompanying drawings illustiate aspects of the
present invention. In such
drawings:
[0048] FIG. 1 is a graph illustrating a y-axis of vector genomes per diploid
genome and an x-axis
of different AAV serotypes AAV3b, AAV3ST, AAVS, and AAV9, as measured in whole
blood, in
accordance with at least one embodiment.
[0049] FIG. 2 is a graph illustrating a y-axis of vector genomes per diploid
genome and an x-axis
of different AAV serotypes AAV3b, AAV3ST, AAVS, and AAV9, as measured in left,
median and
right liver lobes, in accordance with at least one embodiment.
[0050] FIGS. 3A-3B are exemplary plasmids for production of rAAV vectors
useful in the
methods and compositions as disclosed herein. FIG. 3A is an illustration of a
plasmid map of pAAV-
LSPhGAA plasmid for production of a rAAV vector in a producer cell line, e.g.,
a pro-10 cell line, in
accordance with at least one embodiment, where the plasmid comprises a 5' ITR,
LSP, hGAA nucleic
acid sequence, 3' UTR, polyA sequence, and 3' ITR, where the ITRs are from
AAV2. FIG. 3B shows
a more detailed map of the illustration of the plasmid map of FIG. 3A.
[0051] FIGS. 4A-4G are illustrations of exemplary nucleic acid constructs for
a rAAV genome as
disclosed herein that have a targeting peptide, using hGAA as the exemplary
lysosomal protein being
expressed. FIG. 4A shows a nucleic acid construct for a rAAV genome,
comprising a 5' ITR, a Liver
specific promoter (LSP), operatively linked to a heterologous nucleic acid
encoding a secretory signal
peptide (SS), a targeting peptide (TP) and a human GAA (hGAA) polypeptide, and
a 3' ITR. FIG 4B
shows an exemplary nucleic acid construct for a rAAV genome as disclosed
herein, comprising the
same elements as FIG 4A, and additionally comprising at least one polyA signal
3' of the hGAA
polypeptide and 5' of the 3'-ITR. FIG. 4C shows an exemplary nucleic acid
construct for a rAAV
genome as disclosed herein, comprising the same elements as FIG 4B, except
comprising with an
intron sequence 3' of the promoter. FIG. 4D shows an exemplary nucleic acid
construct for a rAAV
genome as disclosed herein, comprising the same elements as FIG 4C, except
comprising a collagen
stability (CS) sequence and/or a 3' UTR sequence located 3' of the hGAA
polypeptide nucleic acid
sequence and before the poly A sequence. FIG. 4E shows an exemplary nucleic
acid construct for a
rAAV genome as disclosed herein, comprising the same elements as FIG 4D,
except also comprising
a nucleic acid encoding a spacer of at least 1 amino acid that is located
between the nucleic acid
encoding the hGAA polypeptide and the nucleic acid encoding the targeting
peptide (TP), e.g., IGF2
targeting peptide. FIG. 4F shows an exemplary nucleic acid construct for a
rAAV genome as
disclosed herein, comprising the same elements as FIG 4E, wherein the promoter
is a liver promoter,
the intron sequence is selected from a MVM or IABB2 intron sequence, the
secretory signal peptide is
selected from any of FN1 signal peptide (e.g., hFN1, ratFN1), a AAT signal
peptide or a hGAA signal
peptide; the targeting peptide is a IGF2 targeting peptide as disclosed
herein, and the at least polyA
sequence is selected from hGHpA or a synPA poly A sequence. FIG. 46 shows an
exemplary
nucleic acid construct for a rAAV genome as disclosed herein, comprising the
same elements as FIG
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4F, except where the IGF2 targeting peptide is a nucleic acid sequence
selected from SEQ ID NO: 2
(IGF2 A2-7), SEQ ID NO: 3 (IGF2 AI-7), or SEQ ID NO: 4 (IGF2 V43M).
[0052] FIG. 5A-5G shows an exemplary nucleic acid constructs for a rAAV
genome. FIG. 5A is a
schematic of exemplary rAAV genome comprising a 5' ITR, a liver specific
promoter, operatively
linked to a nucleic acid encoding a hGAA polypeptide, a polyA sequence (e.g.,
any one or more of
hGHpA, synPA, RBG or SV40 polyA sequences) and a 3' FIR. FIG. 5B is a
schematic of an
exemplary rAAV genome comprising a 5' ITR, a liver specific promoter,
operatively linked to a
nucleic acid encoding a signal secretory peptide (e.g., selected from any of
FN1, AAT or cognate
GAA signal peptide, IL2, mutIL2, IgG), a nucleic acid encoding a human GAA
polypeptide and a
polyA sequence and a 3' ITR_ FIG. 5C is a schematic of an exemplary rAAV
genome comprising a
5' ITR, a liver specific promoter, operatively linked to an intron sequence
(e.g., MVM, SV40 or
HIBB2 intron sequence), a nucleic acid encoding a signal secretory peptide
(e.g., selected from any of
FN1, AAT or cognate GAA signal peptide, IL2, mutIL2, IgG), a nucleic acid
encoding a human GAA
polypeptide and a polyA sequence and a 3' ITR. FIG, 5D is a schematic of a
similar construct to FIG
5C, which includes a collagen stability (CS) sequence or 3' UTR located
between the 3' of the nucleic
acid encoding GAA and the at least one polyA sequence (e.g., hGHpA and/or
synPA polyA
sequence). In some embodiments, the construct comprses both a CS sequence and
a 3 UTR sequence
as disclosed herein. In some embodiments, the CS sequence can be replaced by a
3' UTR sequence as
disclosed herein. In FIGS 5A-5D, exemplary liver specific promoter can be
selected from any of those
disclosed in Table 4 herein, and include, but are not limited to SEQ ID NOs
86, 91-96, or 146-150, or
a sequence with at least 85% sequence identity to SEQ ID NOs: 86, 91-96, or
146-150. FIG. 5E is a
schematic of one embodiment of a AAV vector useful in the methods and
compositions as disclosed
herein for treating Pompe Disease, comprising, flanked between a 5' ITR and a
3' Filt sequence, the
nucleic acid comprising in a 5' to 3' direction: a LSP promoter, a kozak
sequence, a signal sequence
(referred to as leader sequence in FIG 5E), a nucleic acid encoding hGAA and a
poly A sequence. In
some embodiments, the leader sequence can be selected from any of: innate GAA
leader sequence,
IL2 leader sequence (IL2 wt), a modified IL2 leader sequence (IL2 mut) or IgG
leader sequence or
fimctional variants thereof; and the hGAA sequence can be selected from a
consensus hGAA nucleic
acid sequence or a hGAA nucleic acid with at least the H201L mutation, or
other modifications as
disclosed herein (e.g., H199R, R223H). FIG. 5F is a schematic of another
embodiment of a AAV
vector usefid in the methods and compositions as disclosed herein for treating
Pompe Disease,
comprising in a 5' to 3' direction: a liver specific promoter, an intron
sequence, a kozak sequence, a
signal sequence (also referred to as a leader sequence), an IGF2 targeting
peptide sequence (referred
to in FIG. 5F as a "GILT"), a nucleic acid encoding hGAA, optionally a 3' UTR
sequence, and a poly
A sequence, showing that different embodiments, e.g., the promoter can be
selected from any LSP as
disclosed herein, e.g., LSP that have different levels of expression, such as
a High-expression level
LSP (LSP-H), a medium expression level LSP (LSP-M) or low-expressing LSP (LSP-
L), the intron
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sequence can be selected from HBB2, MVM, SV40 and other intron sequences, the
leader sequence
can be selected from any of: innate GAA leader sequence, AAT sequence
(referred to as A1AT in
FIG. 5F), IL2(1-3), IL2 leader sequence (IL2 wt), a modified IL2 leader
sequence (IL2 mut),
fibronectin (FN1, referred to as FBN in FIG. 5F), or IgG leader sequence or
functional variants
thereof; an IGF2 targeting peptide sequence selected from any of the IGF2
targeting peptides
described herein, e.g., WIT IGF2 (SEQ ID NO: 1), A2-7, V43M (SEQ ID NO: 9), A2-
7V43M, or
functional variants thereof, and a hGAA nucleic acid sequence that is codon
optimized as disclosed
herein, e.g., C1-10, which can optionally also comprise at least the H201L
mutation, and/or other
modifications as disclosed herein (e.g., H199R, R223H), and a polyA sequence,
selected from, e.g.,
RBG or SV40 polyA. The LSP designated LSP-H, M-LSP and LSP-L represent liver
specific
promoters that predominantly and preferentially express hGAA in the liver, but
can express hGAA in
one or more other tissues, for example, in the muscle. Such LSPs allows for
expression in the liver for
systemic secretion and uptake by the muscle cells, as well as some expression
in the muscle tissues.
FIG 5G shows schematic of different embodiments of a AAV vector construct
useful in the methods
and compositions as disclosed herein for treating Pompe Disease, where
construct 1 (top panel) shows
a rAAV vector construct comprising in a 5' to 3' direction, a 5' ITR, AAV P5
promoter, liver-specific
promoter (LSP), hGAA nucleic acid sequence, truncated polyA sequence (t-pA),
and 3' ITR; and
Construct 2 (bottom panel) shows an exemplary rAAV vector construct where the
P5 AAV promoter
fragment is removed, the construct comprising in a 5' to 3' direction, a 5'
ITR, liver-specific promoter
(LSP), hGAA nucleic acid sequence, full length polyA sequence (fl-pA), a
terminator sequence in the
antisense orientation and 3' ITR (in the sense orientation).
[0053] FIG. 6 shows an illustration of the Gibson cloning technique to
generate rAAV genomes as
disclosed herein. In particular, a triple ligation is performed to ligate 3
blocks of nucleic acid sequence
together, which can then be cloned into a vector with the promoter, e.g.,
liver specific promoter, and
5' and 3' ITRs to generate the rAAV genome. The Gibson cloning methodology was
used to generate
the following rAAV genomes: SEQ ID NO: 57 (AAT-V43M-wtGAA (deltal-69aa)); SEQ
ID NO: 58
(ratFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA
(deltal-
69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN
lrat- IGFA2-7-
wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1- IGFA2-7-wtGAA (delta 1-69)).
[0054] FIG. 7 shows the generation of an exemplary rAAV genome of SEQ ID NO:
57 comprising
AAT-V43M-wtGAA (deltal-69aa)) using Gibson cloning of nucleic acid sequence
blocks (1, 2 and
3). One of ordinary skill in the art can readily replace the FIR liver
promoter with any of the liver
specific promoters disclosed in Table 4 herein, including but not limited to a
promoter selected from
any of SEQ ID NO: 86, 91-96, 146-150, or a functional variant or functional
fragment thereof. Also
shown in the AAT-V43M-wtGAA (deltal-69aa)) vector is the location a 3 amino
acid (3aa) spacer
nucleic acid sequence (showing the exemplary 3aa sequence "G-A-P" as SEQ ID
NO: 31) which is
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located 3' of the nucleic acid sequence encoding the IGF2(V43M) targeting
peptide and 5'of the
nucleic acid encoding wtGAA(A1-69) enzyme, and a stuffer nucleic acid sequence
(referred to in FIG
8. as a "spacer" sequence) which is located 3' of the polyA sequence and 5' of
the 31TR sequence.
100551 FIG. 8 shows the generation of a rAAV genome of SEQ ID NO: 62
comprising hEN1-
IGF62-7-wtGAA (delta 1-69), using Gibson cloning of nucleic acid sequence
blocks (8, 2 and 3). One
of ordinary skill in the art can readily replace the TTR liver promoter with
any of the liver specific
promoters disclosed in Table 4 herein, including but not limited to a promoter
selected from any of
SEQ ID NO: 86, 91-96, 146-150, or a functional variant or functional fragment
thereof. Also shown
in the hFN1- IGFA2-7-wtGAA (delta 1-69) vector is the location a 3 amino acid
(3aa) spacer nucleic
acid sequence (showing the exemplary 3aa sequence "G-A-1" as SEQ ID NO: 31)
which is located 3'
of the nucleic acid sequence encoding the IGFA2-7targeting peptide and 5'of
the nucleic acid
encoding wtGAA(A1-69) enzyme, and a sniffer nucleic acid sequence (referred to
in FIG 13. as a
"spacer" sequence) which is located 3' of the polyA sequence and 5' of the
3'ITR sequence.
100561 FIGS. 9A-9F shows schematics of exemplary constructs of rAAV genomes
expressing
wild-type GAA. FIG. 9A shows a schematic of exemplary rAAV genome construct of
Candidate
I AAT hIGF2-V43M wtGAA dell-69 Stuffer.V02 (SEQ ID NO: 79). FIG. 9B shows a
schematic
of exemplary rAAV genome construct of Candidate 2_FIBrat_hIGF2-V43M_wtGAA_del
1-
69 Stuffer.V02 (SEQ ID NO: 80). FIG. 9C shows a schematic of exemplary rAAV
genome construct
of Candidate 3_FIBhum_hIGF2-V43M_wtGAA del 1-69_Stuffer.V02 (SEQ ID NO: 81)
FIG. 9D
shows a schematic of exemplary rAAV genome construct of Candidate
4_AAT_GILT_wtGAA_del 1-
69 ________________ Stuffer.V02 (SEQ ID NO: 82). FIG. 9E shows a schematic of
exemplary rAAV genome
construct of Candidate 5_FIBrat_GILT_wtGAA_dell-69_Stuffer.V02 (SEQ ID NO:
83). FIG. 9F
shows a schematic of exemplary rAAV genome construct of Candidate
6 FIBhum GILT wtGAA dell-69 Stuffer.V02 (SEQ ID NO: 84). One of ordinary skill
in the art
can readily replace the FIR liver promoter shown in FIGS 9A-9F for any LPS,
e.g., any liver specific
promoters disclosed in Table 4 herein, including but not limited to a promoter
selected from any of
SEQ ID NO: 86, 91-96, or 146-150. Moreover, FIR promoter can be replaced with
a LSP that can
express the hGAA polypeptide preferentially in the liver and also in at least
one other tissue of
interest, e.g., the muscle, or CNS, and in some embodiments, the FIR promoter
can be replaced with
a LSP that can express the hGAA polypeptide preferentially in the liver and
the muscle and CNS
tissues. In some embodiments, the expressed lysosomal enzyme, e.g., GALA
protein can be configured
as (IAA-fusion protein with a targeting sequence, such as a IGF2 targeting
peptide as disclosed herein
that targets the GALA protein to lysosomes, and/or fused with a signal peptide
(SP), the GALA protein is
expressed by the rAAV genome in the liver, where it is secreted and taken up
by lysosomes of
mammalian cells, in particular muscle cells.
104571 As these are exemplary constructs for illustration purposes only, one
can also readily
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substitute the wtGAA sequence with a codon optimized sequence as disclosed
herein, or a GAA
sequence that has been modified to reduce CpG islands and/or to reduce innate
immunity as disclosed
herein (see FIG. 11B).
[0058] FIG. 10 shows the mean in vivo luciferase expression in mice driven by
exemplary liver-
specific promoters SP0244 and 5P0239. The expression level is shown as the
mean bioluminescence
intensity total flux (in photons per second). Error bars are standard error of
the mean. When animals
are injected with saline only (n=10), no luciferase bioluminescence is
detected. When animals are
injected with a construct comprising luciferase operably linked to the LP1
promoter (n=9), luciferase
bioluminescence is detected. To test the activity of exemplary liver-specific
promoters, animals are
injected with an equivalent construct comprising luciferase operably inked to
the SP0244 promoter
(n=8) and the SP0239 promoter (n=10). Promoters SP0244 and SP0239 showed
higher luciferase
expression in vivo than the control LP1.
[0059] FIGS. 11A-11D shows exemplary modifications to the nucleic acid
sequence encoding the
GAA polypeptide, and the nucleic acid construct to optimize for GAA protein
expression by AAV in
vivo. FIG. 11A shows a schematic of the wild type GAA (wtGAA) nucleotide
sequence operatively
linked to a liver specific promoter as disclosed herein, e.g., a LSP of Table
4, with alternative reading
frames shown by the arrows, and three CpG islands. FIG. 11B shows a schematic
similar to FIG. 11A
that shows in more detail modifications to the nucleic acid sequence encoding
GAA to remove the
CpG islands. FIG. 11C shows modifications to the wtGAA nucleic acid sequence
of SEQ ID NO:
182 that has been modified to (i) reduce the alternative reading frames, (ii)
the number of CpG islands
and (iii) to include modifications for an optimal Kozak sequence. FIG. 11D is
another schematic to
show modifications in the nucleic acid sequence encoding the GAA polypeptide
to reduce the
alternative reading frames, the number of CpG islands and to modifications for
an optimal Kozak
sequence.
[0060] FIG. 12 shows schematics of exemplary rAAV constructs comprising LSP
for expressing
GAA under liver specific promoters. The LSP can be selected from any of the
liver specific promoters
disclosed in Table 4 herein, with or without a s-tuffer sequence.
[0061] FIGS. 13A-13B show GAA expression from construct comprising liver
specific promoters
5P0412 and SP0422 in Huh 7 cells and flEPG2 cells. FIG. 13A shows a western
blot of GAA
expression from construct comprising the liver specific promoter SP0412 (SEQ
ID NO: 91) and
SP0422 (SEQ ID NO: 92) in Huh 7 cells. FIG. 13A shows that expression of hGAA
using promoters
412 (SEQ ID NO: 91) and 422 (SEQ ID NO: 92) leads to significantly higher
expression of hGAA in
Huh7 cells as compared to the expression using the LP1 promoter (SEQ ID NO:
432) which is
referred to as "LSP SS". FIG. 13B shows a western blot of GAA expression from
construct
comprising the liver specific promoter 5P0412 (SEQ ID NO: 91) and 5P0422 (SEQ
ID NO: 92) in
HEPG2 cells. GAA polypeptide was expressed from rAAV generated using the
following plasmids:
LSP NEW (SEQ ID NO: 160), 412 NEW (SEQ ID NO: 159), FIR NEW (SEQ ID NO: 155),
LSP ss
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(AAV with LP-1), 412 TT'R, 422 Stuffer (SEQ ID NO: 158), 422 TIE, 412 Staffer
(SEQ ID NO:
156). FIG. 13B shows that expression of hGAA using promoters 412 (SEQ ID NO:
91) and 422 (SEQ
ID NO: 92) leads to significantly higher expression of hGAA in HepG2 cells as
compared to the
expression using the LP1 promoter (SEQ ID NO: 432) which is referred to as
"LSP SS".
100621 The above described figures illustrate aspects of the invention in at
least one of its
exemplary embodiments, which are further defined in detail in the following
description. Features,
elements, and aspects of the invention that are referenced by the same
numerals in different figures
represent the same, equivalent, or similar features, elements, or aspects, in
accordance with one or
more embodiments.
DETAILED DESCRIPTION
100631 The disclosure described herein generally relates to recombinant AAV
(rAAV) vectors and
constructs for rAAV genomes for gene therapy for delivering a lysosomal
protein, such as a GAA
polypeptide to a subject. In particular, the technology described herein
relates in general to a rAAV
vector, or a rAAV genome for producing a lysosomal protein, e.g., GAA
polypeptide that is expressed
in the liver and effectively targeted to the lysosomes of mammalian cells, for
example, human cardiac
and skeletal muscle cells. For example, the technology relates to a rAAV
vector for transducing liver
cells, where the transduced liver cells secrete the GAA polypeptide, and the
secreted GAA
polypeptide is targeted to lysosomes in skeletal muscle tissue, cardiac muscle
tissue, diaphragm
muscle tissue or a combination thereof
100641 Accordingly, one aspect of the technology described herein provides a
rAAV vector
comprising a rAAV genome that can be used to produce a lysosomal protein,
e.g., GAA or modified
GAA, that is more effectively secreted from cells, e.g., liver cells, and then
targeted to the lysosomes
of mammalian cells, for example, human cardiac and skeletal muscle cells.
100651 hi particular, in some embodiments, the lysosomal
protein, e.g., GAA polypeptide is
expressed by itself. In some embodiments, the lysosomal protein is expressed
as a fusion protein
comprising at least a signal peptide that promotes secretion of the lysosomal
protein, e.g., GAA
polypeptide from the liver. In some embodiments, the GAA polypeptide, or
modified GAA, is
expressed as a fusion protein comprising at least a signal peptide that
promotes secretion of the GAA
polypeptide from the liver, and also a targeting sequence, that allows
effective targeting to lysosomes
in mammalian cells, e.g., muscle cells, for example, human cardiac and
skeletal muscle cells. In some
embodiments, the targeting peptide is a IGF2 targeting peptide a described
herein.
100661
One aspect of the technology
described herein relates to a rAAV vector that comprises a
nucleotide sequence containing inverted terminal repeats (ITRs), a liver
specific promoter, a
heterologous gene, a poly-A tail and potentially other regulator elements for
use to treat a disease,
such as Pompe Disease, and further, for the treatment of Pompe Disease,
wherein the heterologous
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gene is a GAA and wherein the rAAV GAA can be administered to a patient in a
therapeutically
effective dose that is delivered to the appropriate tissue and/or organ for
expression of the
heterologous gene and treatment of the disease.
[0067] One aspect of the technology described herein relates to a rAAV vector
that comprises in its
genome the following in a 5' to 3' direction: 5'- and 3'-AAV inverted terminal
repeats (ITR)
sequences, and located between the 5' and 3' 11Rs, a heterologous nucleic acid
sequence encoding an
alpha-glucosidase (GAA) polypeptide, wherein the heterologous nucleic acid is
operatively linked
to a liver specific promoter, for example, a liver specific promoter disclosed
in Table 4 herein, or a
functional variant thereof. Another aspect of the technology described herein
relates to a rAAV vector
that comprises in its genome the following in a 5' to 3' direction: 5'- and 3'-
AAV inverted terminal
repeats (ITR) sequences, and located between the 5' and 3' ITRs, a
heterologous nucleic acid
sequence encoding a secretory signal peptide (SS), a nucleic acid sequence
encoding an alpha-
glucosidase (GAA) polypeptide, wherein the heterologous nucleic acid is
operatively linked to a
liver specific promoter, for example, a liver specific promoter disclosed in
Table 4 herein, or a
functional variant thereof
[0068] One aspect of the technology described herein relates to a rAAV vector
that comprises in its
genome the following in a 5' to 3' direction: 5'- and 3'-AAV inverted terminal
repeats (ITR)
sequences, and located between the 5' and 3' lilts, a heterologous nucleic
acid sequence encoding a
fusion polypeptide comprising (i) a secretory signal peptide (SS), (ii) an
IGF2 targeting peptide;
and (iii) an alpha-glucosidase (GAA) polypeptide, wherein the heterologous
nucleic acid is
operatively linked to a liver specific promoter, for example, a liver specific
promoter disclosed in
Table 4 herein, or a functional variant thereof
[0069] In all aspects of all embodiments of the technology described herein,
the liver specific
promoter expresses the lysosomal protein, e.g., hGAA polypeptide
preferentially in the liver_ In all
aspects of all embodiments of the technology described herein, the liver
specific promoter expresses
the lysosomal protein e.g., hGAA polypeptide preferentially in the liver and
at least one other tissue of
interest, e.g., the muscle, or CNS, and in some embodiments, the LSP can be
replaced with a LSP that
can express the hGAA polypeptide preferentially in the liver and the muscle
and CNS tissues. In all
aspects of all embodiments of the technology described herein, in some
embodiments where the AAV
vector comprises at least one capsid protein targeting the muscle, the liver
specific promoter can be
replaced with another promoter, e.g., a muscle promoter.
[0070] In some embodiments of the methods and compositions as disclosed
herein, the secretory
signal peptide is selected from any of AAT signal peptide, a fibronectin
signal peptide (FN1), a GAA
signal peptide, or an active fragment thereof having secretory signal
activity.
[0071] In some embodiments, the a rAAV vector described herein is from any
serotype. In some
embodiments, the rAAV vector is a AAV3b serotype, including, but not limited
to, an AAV3b265D
virion, an AAV3b265D549A virion, an AAV3b549A virion, an AAV3bQ263Y virion, or
an
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AAV36SASTG virion (i.e., a virion comprising a AAV3b capsid comprising
Q263A11265
mutations). In some embodiments, the rAAV vector comprises a liver specific
capsid, e.g., a liver
specific capsid selected from XL32 and XL32.1, as disclosed in W02019/241324,
which is
incorporated herein in its entirety by reference. In some embodiments, the
rAAV vector is a
AAVXL32 or AAVXL32.1 as disclosed in W02019/241324, which is incorporated
herein in its
entirety by reference. In some embodiments, the rAAV vector is a rAAV8 vector,
or a haploid rAAV
vector comprising at least one capsid protein from AAV8 (i.e., any one or more
of VP I, VP2 or VP3
is from AAV8 or a chimeric protein thereof). In some embodiments, the AAV
vector comprises a
capsid disclosed in W02019241324A1, or International Patent application
PCT/US2019/036676,
which are incorporated herein in their entirety by reference. In some
embodiments, the AAV vector
comprises a capsid which is encoded by a nucleic acid AAV capsid coding
sequence that is at least
90% identical to a nucleotide sequence of any one of SEQ ID NOs: 1-3 as
disclosed in
W02019241324A1; or (b) a nucleotide sequence encoding any one of SEQ ID NOS:4-
6 as disclosed
in W02019241324A1. In some embodiments, an AAV capsid comprises an amino acid
sequence at
least 90% identical to any one of SEQ ID NOS:4-6 as disclosed in
W02019241324A1, along with
AAV particles comprising an AAV vector genome and the AAV capsid of the
invention. In some
embodiments, the rAAV vector comprises capsid proteins such that the AAV
vector transduces liver
cells, and in some embodiments the rAAV vector comprises the rAAV vector
comprises capsid
proteins such that the AAV vector transduces muscle and liver cells. In such
embodiments, where the
rAAV comprises capsid proteins that enable transduction of muscle cells, the
LSP can be replaced
with another promoter, e.g., a muscle promoter, or promoter that expresses a
protein in liver cells and
muscle cells.
I. Definitions
100721 The following terms are used in the description herein and the appended
claims:
100731 The terms "a," "an," "the" and similar references used in the context
of describing the
present invention (especially in the context of the following claims) are to
be construed to cover both
the singular and the plural, unless otherwise indicated herein or clearly
contradicted by context.
Further, ordinal indicators ¨ such as "first," "second," "third," etc. ¨ for
identified elements are used
to distinguish between the elements, and do not indicate or imply a required
or limited number of such
elements, and do not indicate a particular position or order of such elements
unless otherwise
specifically stated. All methods described herein can be performed in any
suitable order unless
otherwise indicated herein or otherwise clearly contradicted by context. The
use of any and all
examples, or exemplary language (e.g., "such as") provided herein is intended
merely to better
illuminate the present invention and does not pose a limitation on the scope
of the invention otherwise
claimed. No language in the present specification should be construed as
indicating any non-claimed
element essential to the practice of the invention.
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[0074] Furthermore, the term "about," as used herein when referring to a
measurable value such as
an amount of the length of a polynucleotide or polypeptide sequence, dose,
time, temperature, and the
like, is meant to encompass variations oft 20%, 10%, 5%, 1%, 0.5%, or
even 0.1% of the
specified amount.
[0075] Also as used herein, "and/or" refers to and encompasses any and all
possible combinations
of one or more of the associated listed items, as well as the lack of
combinations when interpreted in
the alternative ("or").
[0076] As used herein, the transitional phrase "consisting essentially of
means that the scope of a
claim is to be interpreted to encompass the specified materials or steps
recited in the claim, "and those
that do not materially affect the basic and novel characteristic(s)" of the
claimed invention. See, in re
Hen, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the
original); see also
MPEP 2111.03. Thus, the term "consisting essentially of when used in a claim
of this invention is
not intended to be interpreted to be equivalent to "comprising." Unless the
context indicates
otherwise, it is specifically intended that the various features of the
invention described herein can be
used in any combination.
[0077] Moreover, the present invention also contemplates that in some
embodiments of the
invention, any feature or combination of features set forth herein can be
excluded or omitted.
[0078] To illustrate further, if, for example, the
specification indicates that a particular amino acid
can be selected from A, G, I, Land/or V, this language also indicates that the
amino acid can be
selected from any subset of these amino acid(s) for example A, G, I or L; A,
G, I or V; A or G; only
L; etc. as if each such subcombination is expressly set forth herein.
Moreover, such language also
indicates that one or more of the specified amino acids can be disclaimed
(e.g., by negative proviso).
For example, in particular embodiments the amino acid is not A, Cl or I; is
not A; is not Cl or V; etc. as
if each such possible disclaimer is expressly set forth herein.
[0079] The term "parvovirus" as used herein encompasses the family
Parvoviridae, including
autonomously replicating parvoviruses and dependoviruses. The autonomous
parvoviruses include
members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and
Contravirus. Exemplary
autonomous parvoviruses include, but are not limited to, minute virus of
mouse, bovine parvovirus,
canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline
parvovirus, goose
parvovirus, HI parvovirus, Muscovy duck parvovirus, B19 virus, and any other
autonomous
parvovirus now known or later discovered. Other autonomous parvoviruses are
known to those skilled
in the art. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter
69 (4th ed.,
Lippincott-Raven Publishers).
[0080] As used herein, the term "adeno-associated virus" (AAV), includes but
is not limited to,
AAV type I, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4,
AAV type 5, AAV
type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian
AAV, bovine
AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later
discovered.
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See, e.g., BERNARD N. FIELDS et at, VIROLOGY, volume 2, chapter 69 (4th ed.,
Lippincott-
Raven Publishers). A number of relatively new AAV serotypes and clades have
been identified (see,
e.g., Gao et al., (2004)1 Virology 78:6381-6388; Moris et al., (2004) Virology
33-175- 383); and
also Table 1 as disclosed in U.S. Provisional Application 62,937,556, filed on
November 19, 2019 and
Table tin International Applications W02020/102645, and W02020/102667, each of
which is
incorporated herein in their entirety.
100811 The genomic sequences of various serotypes of AAV and the autonomous
parvoviruses, as
well as the sequences of the native inverted terminal repeats (flits), Rep
proteins, and capsid subunits
are known in the art. Such sequences may be found in the literature or in
public databases such as
GenBank. See, e.g., GenBank Accession Numbers NC_002077, NC_001401, NC_001729,
NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152,
NC 006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901,
J02275,
X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540,
AF513851,
AF513852, AY530579; the disclosures of which are incorporated by reference
herein for teaching
parvovirus and AAV nucleic acid and amino acid sequences. See also, e.g.,
Srivistava et al., (1983) J
Virology 45:555; Chiarini et al,, (1998)1 Virology 71:6823; Chiarini et al.,
(1999).1 Virology
73:1309; Bantel-Schaal et al., (1999)1 Virology 73:939; Xiao et al., (1999)1
Virology 73:3994;
Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986)1 Viral.
58:921; Gao et al., (2002)
Proc. Nat Acad. Sci. USA 99:11854; Morris et al., (2004) Virology 33-:375-
383; international patent
publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No.
6,156,303; the
disclosures of which are incorporated by reference herein for teaching
parvovirus and AAV nucleic
acid and amino acid sequences. See also Table 1 and Table 5 disclosed in
62,937,556, filed on
November 19, 2019 or Table 1 as disclosed in International Applications
W02020/102645, and
W02020/102667, each of which is incorporated herein in their entirety. The
capsid structures of
autonomous parvoviruses and AAV are described in more detail in BERNARD N.
FIELDS et at,
VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).
See also,
description of the crystal structure of AAV2 (Xie et at., (2002) Proc. Nat
Acad. Sc!. 99:10405-10),
AAV4 (Padron et al., (2005)1 Viral 79: 5047-58), AAV5 (Walters et al., (2004)1
Viral 78: 3361-
71) and CPV (Xie et al., (1996)1 Mat Blot 6:497-520 and Tsao et al., (1991)
Science 251: 1456-64).
100821 The term "tropism" as used herein refers to preferential entry of the
virus into certain cells
or tissues, optionally followed by expression (e.g., transcription and,
optionally, translation) of a
sequence(s) carried by the viral genome in the cell, e.g., for a recombinant
virus, expression of a
heterologous nucleic acid(s) of interest.
100831 As used here, "systemic tropism" and "systemic transduction" (and
equivalent terms)
indicate that the virus capsid or virus vector of the invention exhibits
tropism for and/or transduces
tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver,
kidney and/or pancreas).
In embodiments of the invention, systemic transduction of the central nervous
system (e.g., brain,
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neuronal cells, etc.) is observed. In other embodiments, systemic transduction
of cardiac muscle
tissues is achieved.
[0084] As used herein, "selective tropism" or "specific tropism" means
delivery of virus vectors to
and/or specific transduction of certain target cells and/or certain tissues.
[0085] Unless indicated otherwise, "efficient transduction" or "efficient
tropism," or similar terms,
can be determined by reference to a suitable control (e.g., at least about
50%, 600%, 70%, 80%, 850%,
90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more
of the
transduction or tropism, respectively, of the control). In particular
embodiments, the virus vector
efficiently transduces or has efficient tropism for liver cells and muscle
cells. Suitable controls will
depend on a variety of factors including the desired tropism and/or
transduction profile.
[0086] Similarly, it can be determined if a virus "does not efficiently
transduce" or "does not have
efficient tropism" for a target tissue, or similar terms, by reference to a
suitable control. In particular
embodiments, the virus vector does not efficiently transduce (i.e., has does
not have efficient tropism)
for kidney, gonads and/or germ cells. In particular embodiments, transduction
(e.g., undesirable
transduction) of tissue(s) (e.g., kidney) is 20% or less, 10% or less, 5% or
less, 1% or less, 0.1% or
less of the level of transduction of the desired target tissue(s) (e.g.,
liver, skeletal muscle, diaphragm
muscle, cardiac muscle and/or cells of the central nervous system).
[0087] In some embodiments of this invention, an AAV particle comprising a
capsid of this
invention can demonstrate multiple phenotypes of efficient transduction of 30
certain tissues/cells and
very low levels of transduction (e.g., reduced transduction) for certain
tissues/cells, the transduction of
which is not desirable.
[0088] As used herein, the term "polypeptide" encompasses both peptides and
proteins, unless
indicated otherwise.
[0089] A "polynucleotide" is a sequence of nucleotide bases, and may be RNA,
DNA or DNA-
RNA hybrid sequences (including both naturally occurring and non-naturally
occurring nucleotides),
but in representative embodiments are either single or double stranded DNA
sequences.
[0090] The terms "beterologotis nucleotide sequence" and "heterologous nucleic
acid molecule"
are used interchangeably herein and refer to a nucleic acid sequence that is
not naturally occurring in
the virus. Generally, the heterologous nucleic acid molecule or hetcrologous
nucleotide sequence
comprises an open reading frame that encodes a polypeptide and/or 110/1traild
ated RNA of interest
(e.g., for delivery to a cell and/or subject).
[0091] A "chimeric nucleic acid" comprises two or more nucleic acid sequences
covalently linked
together to encode a fusion polypeptide. The nucleic acids may be DNA, RNA, or
a hybrid thereof.
100921 The term "fusion polypeptide" comprises two or more polypeptides
covalently linked
together, typically by peptide bonding.
100931 As used herein, an "isolated" polynucleotide (e.g., an "isolated DNA"
or an "isolated
RNA") means a polynucleotide at least partially separated from at least some
of the other components
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of the naturally occurring organism or virus, for example; the cell or viral
structural components or
other polypeptides or nucleic acids commonly found associated with the
polynucleotide. In
representative embodiments an "isolated" nucleotide is enriched by at least
about 10-fold, 100'-fold,
1000-fold, 10,000-fold or more as compared with the starting material.
[0094] Likewise, an "isolated" polypeptide means a polypeptide that is at
least partially separated
from at least some of the other components of the naturally occurring organism
or virus, for example,
the cell or viral structural components or other polypeptides or nucleic acids
commonly found
associated with the polypeptide. In representative embodiments an "isolated"
polypeptide is enriched
by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as
compared with the starting
material.
[0095] An "isolated cell" refers to a cell that is separated from other
components with which it is
normally associated in its natural state. For example, an isolated cell can be
a cell in culture medium
and/or a cell in a pharmaceutically acceptable carrier of this invention.
Thus, an isolated cell can be
delivered to and/or introduced into a subject. In some embodiments, an
isolated cell can be a cell that
is removed from a subject and manipulated as described herein ex vivo and then
returned to the
subject.
[0096] A population of virions can be generated by any of the methods
described herein. In one
embodiment, the population is at least 101 virions. In one embodiment, the
population is at least 102
virions, at least 103, virions, at least 104 virions, at least 105 virions, at
least 106 virions, at least 107
virions, at least 108 virions, at least 109 virions, at least 1010 virions, at
least 1011 virions, at least
1012 virions, at least 1013 virions, at least 1014 virions, at least 1015
virions, at least 1016 virions, or
at least 1017 virions. A population of virions can be heterogeneous or can be
homogeneous (e.g.,
substantially homogeneous or completely homogeneous).
[0097] A "substantially homogeneous population" as the term is used herein,
refers to a population of
virions that are mostly identical, with few to no contaminant virions (those
that are not identical)
therein. A substantially homogeneous population is at least 90% of identical
virions (e.g., the desired
virion), and can be at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, at least 99,5%, at least 99.9% of
identical virions.
[0098] A population of virions that is completely homogeneous contains only
identical virions.
[0099] As used herein, by "isolate" or "purify" (or grammatical equivalents) a
virus vector or virus
particle or population of virus particles, it is meant that the virus vector
or virus particle or population
of virus particles is at least partially separated from at least some of the
other components in the
starting material. In representative embodiments an "isolated" or "purified"
virus vector or virus
particle or population of virus particles is enriched by at least about 10-
fold, 100-fold, 1000-fold,
10,000-fold or more as compared with the starting material,
[00100] Unless indicated otherwise, "efficient transduction" or "efficient
tropism," or similar terms,
can be determined by reference to a suitable control (e.g., at least about
10%, 15%, 20%, 25%, 30%,
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35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%,
150%,
175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or
tropism, respectively,
of the control). In particular embodiments, the virus vector efficiently
transduces or has efficient
tropism for neuronal cells and cardiomyocytes. Suitable controls will depend
on a variety of factors
including the desired tropism and/or transduction profile.
[00101] A "therapeutic polypeptide" is a polypeptide that can alleviate,
reduce, prevent, delay
and/or stabilize symptoms that result from an absence or defect in a protein
in a cell or subject and/or
is a polypeptide that otherwise confers a benefit to a subject, e.g., enzyme
replacement to reduce or
eliminate symptoms of a disease, or improvement in transplant survivability or
induction of an
immune response.
[00102] The terms "heterologous nucleotide sequence" and "heterologous nucleic
acid molecule"
are used interchangeably herein and refer to a nucleic acid sequence that is
not naturally occurring in
the virus. Generally, the heterologous nucleic acid molecule or heterologous
nucleotide sequence
comprises an open reading frame that encodes a polypeptide and/or
nontranslated RNA of interest
(e.g., for delivery to a cell and/or subject), for example the GAA
polypeptide.
[00103] As used herein, the terms "virus vector," "vector" or "gene delivery
vector" refer to a virus
AAV) particle that functions as a nucleic acid delivery vehicle, and which
comprises the vector
genome (e.g., viral DNA [vDNA1) packaged within a virion. Alternatively, in
some contexts, the
term "vector" may be used to refer to the vector genome/vDNA alone.
[00104] An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA)
that
comprises one or more heterologous nucleic acid sequences. rAAV vectors
generally require only The
inverted terminal repeat(s) (TR(s)) in cis to generate virus. All other viral
sequences are dispensable
and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbial.
Immunol. 158:97).
Typically, the rAAV vector genome will only retain the one or more TR sequence
so as to maximize
the size of the transgene that can be efficiently packaged by the vector. The
structural and non-
structural protein coding sequences may be provided in trans (e.g., from a
vector, such as a plasmid,
or by stably integrating the sequences into a packaging cell). In embodiments
of the invention the
rAAV vector genome comprises at least one !TR sequence (e.g., AAV TR
sequence), optionally two
ITRs (e.g., two AAV TRs), which typically will be at the 5' and 3' ends of the
vector genome and
flank the heterologous nucleic acid, but need not be contiguous thereto. The
TRs can be the same or
different from each other.
1001051 The term "terminal repeat" or "'TR" includes any viral terminal repeat
or synthetic sequence
that forms a hairpin structure and functions as an inverted terminal repeat
(La, an ITR that mediates
the desired functions such as replication, virus packaging, integration and/or
provirus rescue, and the
like). The TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR
sequence such as
those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus
(MVIVI), human
parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin
that serves as the origin
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of SV40 replication) can be used as a TR, which can further be modified by
truncation, substitution,
deletion, insertion and/or addition. Further, the TR can be partially or
completely synthetic, such as
the "double-D sequence" as described in United States Patent No. 5,478,745 to
Samulski et at
1001061 An "AAV terminal repeat" or "AAV TR," including an "AAV inverted
terminal repeat" or
"AAV ITR" may be from any AAV, including but not limited to serotypes 1, 2, 3,
4, 5, 6, 7, 8, 9, 10,
11 or 12 or any other AAV now known or later discovered. An AAV terminal
repeat need not have
the native terminal repeat sequence (e.g., a native AAV TR. or AAV ITR
sequence may be altered by
insertion, deletion, truncation and/or missense mutations), as long as the
terminal repeat mediates the
desired functions, e.g., replication, virus packaging, integration, and/or
provirus rescue, and the like.
1001071 AAV proteins VP I, VP2 and VP3 are capsid proteins that interact
together to form an AAV
capsid of an icosahedral symmetry. VP! .5 is an AAV capsid protein described
in US Publication No.
2014/0037585.
1001081 The virus vectors of the invention can further be "targeted" virus
vectors (e.g., having a
directed tropism) and/or a "hybrid" parvovirus (Le., in which the viral TRs
and viral capsid are from
different parvoviruses) as described in international patent publication WO
00/28004 and Chao et al.,
(2000) _Molecular Therapy 2:619.
110*1091 The virus vectors of the invention can further be duplexed parvovirus
particles as described
in international patent publication WO 01/92551 (the disclosure of which is
incorporated herein by
reference in its entirety). Thus, in some embodiments, double stranded
(duplex) genomes can be
packaged into the virus c,apsids of the invention.
1001101 Further, the viral capsid or genomic elements can contain other
modifications, including
insertions, deletions and/or substitutions.
1001111 A "chimeric' capsid protein as used herein means an AAV capsid protein
(e.g., any one or
more of VP!, VP2 or VP3) that has been modified by substitutions in one or
more (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the
capsid protein relative to wild
type, as well as insertions and/or deletions of one or more (e.g., 2, 3, 4, 5,
6, 7, 8, 9, 10, etc.) amino
acid residues in the amino acid sequence relative to wild type. In some
embodiments, complete or
partial domains, functional regions, epitopes, etc., from one AAV serotype can
replace the
corresponding wild type domain, functional region, epitope, etc. of a
different AAV serotype, in any
combination, to produce a chimeric capsid protein of this invention.
Production of a chimeric capsid
protein can be canied out according to protocols well known in the art and a
significant number of
chimeric capsid proteins are described in the literature as well as herein
that can be included in the
capsid of this invention.
1001121 As used herein, the term "haploid AAV" shall mean that AAV as
described in International
Application W02018/170310, or US Application US2018/037149, which are
incorporated herein in
their entirety by reference. In some embodiments, a population of virions is a
haploid AAV
population where a virion particle can be constructed wherein at least one
viral protein from the group
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consisting of AAV capsid proteins, VP!, VP2 and VP3, is different from at
least one of the other viral
proteins, required to form the virion particle capable of encapsulating an AAV
genome. For each viral
protein present (VP!, VP2, and/or VP3), that protein is the same type (e.g.,
all AAV2 VP!)! In one
instance, at least one of the viral proteins is a chimeric viral protein and
at least one of the other two
viral proteins is not a chimeric. In one embodiment VP! and VP2 are chimeric
and only VP3 is non-
chimeric. For example, only the viral particle composed of VP1NP2 from the
chimeric AAV2/8 (the
N-terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2;
or only the
chimeric VP1/VP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from
AAV2 without
mutation of VP3 start codon) paired with only VP3 from AAV2. In another
embodiment only VP3 is
chimeric and VP! and VP2 are non-chimeric. In another embodiment at least one
of the viral proteins
is from a completely different serotype. For example, only the chimeric
VP!/VP2 28m-2P3 paired
with VP3 from only AAV3. In another example, no chimeric is present.
100113] The term a "hybrid" AAV vector or parvovirus refers to a rAAV vector
where the viral TRs
or ITRs and viral capsid are from different parvoviruses. Hybrid vectors are
described in international
patent publication WO 00/28004 and Chao etal., (2000) Molecular Therapy 2:619.
For example, a
hybrid AAV vector typically comprises the adenovirus 5' and 3' cis ITR
sequences sufficient for
adenovirus replication and packaging (i.e., the adenovirus terminal repeats
and PAC sequence).
1001141 The term "polyploid AAV" refers to a AAV vector which is composed of
capsids from two
or more AAV serotypes, e.g., and can take advantages from individual serotypes
for higher
transduction but not in certain embodiments eliminate the tropism from the
parents.
1001151 The term "GAA" or "GAA polypeptide," as used herein, encompasses
mature (76 or -67
kDa) and precursor (e.g., -110 kDa) GAA as well as modified (e.g., truncated
or mutated by
insertion(s), deletion(s) and/or substitution(s)) GAA proteins or fragments
thereof that retain
biological function (i.e., have at least one biological activity of the native
GAA protein, e.g., can
hydrolyze glycogen, as defined above) and GAA variants (e.g., GAA II as
described by Kunita et al.,
(1997) Biochemica et Biophysica Ada 1362:269; GAA polymorphisms and SNPs are
described by
Hirschhorn, R. and Reuser, A. J. (2001) in The Metabolic and Molecular Basis
for Inherited Disease
(Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, if Eds.), pp. 3389-3419,
McGraw-Hill, New
York, see pages 3403-3405; each incorporated herein by reference in its
entirety). Any GAA coding
sequence known in the art may be used, for example, see the coding sequences
of FIGS. 8 and 9;
GenBank Accession number NM 00152 and Hoefsloot et al., (1988) EMBO J. 7:1697
and Van Hove
et al., (1996) Proc. Natl. Acad. Sci. USA 93:65 (human), GenBank Accession
number NM_008064
(mouse), and Kunita et al., (1997) Biochemica et Biophysics Acta 1362:269
(quail); the disclosures of
which are incorporated herein by reference for their teachings of GAA coding
and noncoding
sequences.
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1001161The terms "cation-independent mannose-6-phosphate receptor (CI-MPR),"
"M6P/IGF-II
receptor," "CI-MPRJIGF-II receptor," "IGF-II receptor" or "IGF2 Receptor," or
abbreviations thereof,
are used interchangeably herein, referring to the cellular receptor which
binds both M6P and IGF-II.
1001171 The term "targeting peptide" is also referred to as a' argeting
sequence" as used herein is
intended to refer to a peptide that targets a particular subcellular
compartment, for example, a
mammalian lysosome. A targeting peptide encompassed for use herein is a
lysosome targeting peptide
that is marmose-6-phosphate-independent. An exemplary targeting sequence is an
IGF2 targeting
peptide as disclosed herein.
1001181 The term "IGF2 sequence" is used in conjunction with "IGF2 targeting
sequence" or "IGF2
leader sequence" and "IGF2 targeting peptide" are used interchangeably herein
and refer to a
sequence of the IGF2 polypeptide that binds to the CI-MBR on the surface of
the cell. In particular,
the IGF2 sequence is a peptide that comprises a part of the IGF2 uptake
sequence of SEQ ID NO: 5,
or comprises a modification in amino acid of SEQ ID NO:5. An IGF2 targeting
peptide refers to a
peptide sequence that binds to a receptor domain consisting essentially of
repeats 11-12, repeat 11 or
amino acids 1508-1566 of the human cation-independent mannose-6-phosphate
receptor (CI-MPR or
CA-M6P receptor).
1001191 The term "leader sequence" is used interchangeably herein with the
term "secretory signal
sequence" or "signal sequence" or "signal peptide" or variations thereof, and
intended to refer to
amino acid sequences that function to enhance (as defined above) secretion of
an operably linked
polypeptide, (e.g., a GAA peptide or IGF2-GAA fusion protein) from the cell as
compared with the
level of secretion seen with the native polypeptide. As defined above, by
"enhanced" secretion, it is
meant that the relative proportion of lysosomal polypeptide synthesized by the
cell that is secreted
from the cell is increased; it is not necessary that the absolute amount of
secreted protein is also
increased. In particular embodiments of the invention, essentially all (i.e.,
at least 95%, 97%, 98%,
99% or more) of the GAA-polypeptide is secreted. It is not necessary, however,
that essentially all or
even most of the GAA polypeptide is secreted, as long as the level of
secretion is enhanced as
compared with the native GAA polypeptide. Exemplary leader sequences include,
but are not limited
to the innate GAA leader sequence (also referred to cognate GAA leader
sequence), AAT sequence,
IL2(1-3), IL2 leader sequence (IL2 wt), a modified IL2 leader sequence (IL2
mut), fibronectin (FN1,
also referred to as FBN), or IgG leader sequence or functional variants
thereof, as disclosed herein.
1001201 As used herein, the term "amino acid" encompasses any naturally
occurring amino acid,
modified forms thereof, and synthetic amino acids. Naturally occurring,
levorotatory (L-) amino acids
are disclosed in Table 2 of US Publication 2018/0371496, which is incorporated
herein in its entirety.
Alternatively, the amino acid can be a modified amino acid residue
(nonlimiting examples are shown
in Table 4 of US Publication of US Publication 2018/0371496) and/or can be an
amino acid that is
modified by post-translation modification (e.g., acetylation, amidation,
formylation, hydroxylation,
methylation, phosphorylation or sulfatation). Further, the non-naturally
occurring amino acid can be
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an "unnatural' amino acid as described by Wang et al., Amm Rev Biophys Biomol
Stnict. 35:225-49
(2006). These unnatural amino acids can advantageously be used to chemically
link molecules of
interest to the AAV capsid protein.
1001211 To illustrate further, if, for example, the specification indicates
that a particular amino acid
can be selected from A, G, I, L and/or V, this language also indicates that
the amino acid can be
selected from any subset of these amino acid(s) for example A, G, I or L; A,
G, I or V; A or G; only
L; etc. as if each such subcombination is expressly set forth herein.
Moreover, such language also
indicates that one or more of the specified amino acids can be disclaimed
(e.g., by negative proviso).
For example, in particular embodiments the amino acid is not A, G or I; is not
A; is not G or V; etc. as
if each such possible disclaimer is expressly set forth herein.
1001221The term "cis-regulatory element" or "CRE", is a term well-known to the
skilled person, and
means a nucleic acid sequence such as an enhancer, promoter, insulator, or
silencer, that can regulate
or modulate the transcription of a neighboring gene (i.e. in cis). CREs are
found in the vicinity of the
genes that they regulate. CREs typically regulate gene transcription by
binding to transcription factors
(TFs), i.e. they include TF binding site (T11:3S). A single TF may bind to
many CREs, and hence
control the expression of many genes (pleiotropy). CREs are usually, but not
always, located upstream
of the transcription start site (TSS) of the gene that they regulate.
"Enhancers" are CREs that enhance
(i.e. upregulate) the transcription of genes that they are operably associated
with, and can be found
upstream, downstream, and even within the introns of the gene that they
regulate. Multiple enhancers
can act in a coordinated fashion to regulate transcription of one gene.
"Silencers" in this context
relates to CREs that bind TFs called repressors, which act to prevent or
downregulate transcription of
a gene. The term "silencer" can also refer to a region in the 3' untranslated
region of messenger RNA,
that bind proteins which suppress translation of that mRNA molecule, but this
usage is distinct from
its use in describing a CRE. Generally, the CREs of the present invention are
liver-specific enhancers
(often referred to as liver-specific CREs, or liver-specific CRE enhancers, or
suchlike). In the present
context, it is preferred that the CRE is located 1500 nucleotides or less from
the transcription start site
(TSS), more preferably 1000 nucleotides or less from the TSS, more preferably
500 nucleotides or
less from the TSS, and suitably 250, 200, 150, or 100 nucleotides or less from
the TSS. CREs of the
present invention are preferably comparatively short in length, preferably 100
nucleotides or less in
length, for example they may be 90, 80, 70, 60 nucleotides or less in length.
1001231The term "cis-regulatory module" or "CR_M" means a fimctional module
made up of two or
more CREs; in the present invention the CREs are typically liver-specific
enhancers. Thus, in the
present application a CRNI typically comprises a plurality of liver-specific
enhancer CREs. Typically,
the multiple CREs within the CRNI act together (e.g. additively or
synergistically) to enhance the
transcription of a gene that the CRNI is operably associated with. There is
conservable scope to
shuffle (i.e. reorder), invert (i.e. reverse orientation), and alter spacing
in CREs within a CRM.
Accordingly, functional variants of CRNIs of the present invention include
variants of the referenced
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CRMs wherein CREs within them have been shuffled and/or inverted, and/or the
spacing between
CREs has been altered.
[00124] As used herein, the phrase "promoter" refers to a region of DNA that
generally is located
upstream of a nucleic acid sequence to be transcribed that is needed for
transcription to occur, i.e.
which initiates transcription. Promoters permit the proper activation or
repression of transcription of a
coding sequence under their control. A promoter typically contains specific
sequences that are
recognized and bound by plurality of TFs. TFs bind to the promoter sequences
and result in the
recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding
region of the gene.
A great many promoters are known in the art.
1001251The term "synthetic promote?' as used herein relates to a promoter that
does not occur in
nature. In the present context it typically comprises a synthetic CRE and/or
CRM of the present
invention operably linked to a minimal (or core) promoter or liver-specific
proximal promoter. The
CREs and/or CRMs of the present invention serve to enhance liver-specific
transcription of a gene
operably linked to the promoter. Parts of the synthetic promoter may be
naturally occurring (e.g. the
minimal promoter or one or more CREs in the promoter), but the synthetic
promoter as a complete
entity is not naturally occurring,
1001261 As used herein, "minimal promote?' (also
known as the "core promoter") refers to a
short DNA segment which is inactive or largely inactive by itself, but can
mediate transcription when
combined with other transcription regulatory elements. Minimum promoter
sequence can be derived
from various different sources, including prokaryotic and eukaryotic genes.
Examples of minimal
promoters are discussed above, and include the dopamine beta-hydroxylase gene
minimum promoter,
cytomegalovirus (CMV) immediate early gene minimum promoter (CMV-MP), and the
herpes
thymidine kinase minimal promoter (MinTK). A minimal promoter typically
comprises the
transcription start site (TS5) and elements directly upstream, a binding site
for RNA polymerase II,
and general transcription factor binding sites (often a TATA box).
[00127] As used herein, "proximal promoter" relates
to the minimal promoter plus the
proximal sequence upstream of the gene that tends to contain primary
regulatory elements. It often
extends approximately 250 base pairs upstream of the TSS, and includes
specific TFBS. In the
present case, the proximal promoter is suitably a naturally occurring liver-
specific proximal promoter
that can be combined with one or more CREs or CRMs of the present invention.
However, the
proximal promoter can be synthetic.
1001281A "functional variant" of a cis-regulatory element (CRE), cis-
regulatory module (CRM),
promoter or other nucleic acid sequence in the context of the present
invention is a variant of a
reference sequence that retains the ability to function in the same way as the
reference sequence, e.g.
as a liver-specific cis-regulatory enhancer element, liver-specific cis-
regulatory module or liver-
specific promoter. Alternative terms for such functional variants include
"biological equivalents" or
"equivalents".
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1001291It will be appreciated that the ability of a given cis-regulatory
element to function as a liver-
specific enhancer is determined principally by the ability of the sequence to
bind the same liver-
specific transcription factors (TFs) that bind to the reference sequence.
Accordingly, in most cases, a
functional variant of a cis-regulatory element will contain TFBS for the same
TFs as the reference cis-
regulatory element. It is preferred, but not essential, that the transcription
factor binding site (TIES)
of a functional variant are in the same relative positions (i.e. order) as the
reference cis-regulatory
element. It is also preferred, but not essential, that the TFBS of a
functional variant are in the same
orientation as the reference sequence (it will be noted that TFBS can in some
cases be present in
reverse orientation, e.g. as the reverse complement vis-a-vis the sequence in
the reference sequence).
It is also preferred, but not essential, that the TFBS of a functional variant
are on the same strand as
the reference sequence. Thus, in preferred embodiments, the functional variant
comprises TFBS for
the same TFs, in the same order, in the same orientation and on the same
strand as the reference
sequence. It will also be appreciated that the sequences lying between TFBS
(referred to in some
cases as spacer sequences, or suchlike) are of less consequence to the
function of the cis-regulatory
element. Such sequences can typically be varied considerably, and their
lengths can be altered.
However, in preferred embodiments the spacing (i.e. the distance between
adjacent TFBS) is
substantially the same (e.g. it does not vary by more than 20, preferably by
not more than 10%, more
preferably it is the same) in a functional variant as it is in the reference
sequence. It will be apparent
that in some cases a functional variant of a cis-regulatory enhancer element
can be present in the
reverse orientation, e.g. it can be the reverse complement of a cis-regulatory
enhancer element as
described above, or a variant thereof
[00130] Levels of sequence identity between a fiuictional variant and the
reference sequence can also
be an indicator or retained functionality. High levels of sequence identity in
the TFBS of the cis-
regulatory element is of generally higher importance than sequence identity in
the spacer sequences
(where there is little or no requirement for any conservation of sequence).
However, it will be
appreciated that even within the TFBS, a considerable degree of sequence
variation can be
accommodated, given that the sequence of a functional TFBS does not need to
exactly match the
consensus sequence.
[00131] The ability of one or more TFs to bind to a TFBS in a given functional
variant can determined
by any relevant means known in the art, including, but not limited to,
electromobility shift assays
(EMSA), binding assays, chromatin immunoprecipitation (ChIP), and ChIP-
sequencing (ChIP-seq).
In a preferred embodiment the ability of one or more TFs to bind a given
functional variant is
determined by EMSA. Methods of performing EMSA are well-known in the art.
Suitable approaches
are described in Sambrook et al. cited above. Many relevant articles
describing this procedure are
available, e.g. Hellman and Fried, Nat Protoc. 2007; 2(8): 1849-1861.
[00132] The terms "liver-specific" or "liver-specific expression" when in
reference to a promoter
refers to the ability of a cis-regulatory element, cis-regulatory module or
promoter to enhance or drive
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expression of a gene in the liver (or in liver-derived cells) in a
preferential or predominant manner as
compared to other tissues (e.g. spleen, muscle, heart, lung, and brain).
Expression of the gene can be
in the form of mRNA or protein. In some embodiments, liver-specific expression
is such that there is
negligible expression in other (i.e. non-liver) tissues or cells, i.e.
expression is highly liver-specific. In
some embodiments, while a liver-specific promoter drives expression
preferentially in the liver, it can
also drive expression of the gene in another tissue of interest at a lower
level, e.g., muscle.
1001331The ability of a cis-regulatory element to function as a liver-specific
cis-regulatory enhancer
element can be readily assessed by the skilled person. The skilled person can
thus easily determine
whether any variant of the specific cis-regulatory elements recited above
remains functional (i.e. it is a
functional variant as defined above). For example, any given cis-regulatory
element to be assessed
can be operably linked to a minimal promoter (e.g. positioned upstream of CMV-
MP) and the ability
of the cis-regulatory element to drive liver-specific expression of a gene
(typically a reporter gene) is
measured. Alternatively, a variant of a cis-regulatory enhancer element can be
substituted into a
synthetic liver-specific promoter in place of a reference cis-regulatory
enhancer element, and the
effects on liver-specific expression driven by said modified promoter can be
determined and
compared to the unmodified form. Similarly, the ability of a cis-regulatory
module or promoter to
drive liver-specific expression can be readily assessed by the skilled person
(e.g. as described in the
examples below). Expression levels of a gene driven by a variant of a
reference promoter can be
compared to the expression levels driven by the reference sequence. In some
embodiments, where
liver-specific expression levels driven by a variant promoter are at least
50%, at least 60%, at least
70%, at least 80%, at least 90%, or at least 100% of the expression levels
driven by the reference
promoter, it can be said that the variant remains functional. Suitable nucleic
acid constructs and
reporter assays to assess liver-specific expression enhancement can easily be
constructed, and the
examples set out below give suitable methodologies.
1001341Liver-specificity can be identified wherein the expression of a gene
(e.g. a therapeutic or
reporter gene) occurs preferentially or predominantly in liver-derived cells.
Preferential or
predominant expression can be defined, for example, where the level of
expression is significantly
greater in liver-derived cells than in other types of cells (i.e. non-liver-
derived cells). For example,
expression in liver-derived cells is suitably at least 5-fold higher than non-
liver cells, preferably at
least 10-fold higher than non-liver cells, and it may be 50-fold higher or
more in some case& For
convenience, liver-specific expression can suitably be demonstrated via a
comparison of expression
levels in a hepatic cell line (es. liver-derived cell line such as Huh7 and/or
HepG2 cells) or liver
primary cells, compared with expression levels in a kidney-derived cell line
(e.g. HEK-293), a
cervical tissue-derived cell line (e.g. HeLa) and/or a lung-derived cell line
(e.g. A549).
101)1351The synthetic liver-specific promoters of the present invention are
preferably suitable for
promoting expression in the liver of a subject, e.g. driving liver-specific
expression of a transgene,
preferably a therapeutic transgene. In some embodiments, the liver-specific
promoters of the
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invention are suitable for promoting liver-specific transgene expression at a
level at least 1.5-fold
greater than the LP1 promoter of SEQ ID NO: 432, preferably 2-fold greater
than the LP1 promoter,
more preferably 3-fold greater than the LP1 promoter, and yet more preferably
5-fold greater than the
LP1 promoter (SEQ ID NO: 432). Such expression is suitably determined in liver-
derived cells, e.g.
in Huh7, and/or HepG2 cells or primary liver cells (suitably primary human
hepatocytes). In some
embodiments, the synthetic liver-specific promoters of the present invention
are suitable for
promoting gene expression at a level of at least 1.5-fold less than an LP1
promoter (SEQ ID NO: 432)
in non-liver-derived cells (e.g. HEK-293, HeLa, and/or A549 cells).
1001361Preferred synthetic liver-specific promoters of the present invention
am suitable for
promoting liver-specific transgene expression and have an activity in liver
cells which is at least 15%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%,
300%, 350%
or 400% of the activity of the TBG promoter (SEQ ID NO: 435).
1001371The synthetic liver-specific promoters of the present invention are
preferably suitable for
promoting liver-specific expression at a level at least 1.5-fold greater than
a CMV-IE promoter of
SEQ ID NO: 433 in liver-derived cells, preferably at least 2-fold greater than
a CMV promoter in
liver-derived cells (e.g. HEK-293, HeLa, and/or A549 cells). The synthetic
liver specific promoters
disclosed herein can be LSP-H, LSP-M and LSP-L promoters, referring to high,
medium and low
expression in the liver, and in some embodiments, the LSP-H, LSP-M and LSP-L
can preferentially or
predominantly express a protein in the liver, but can also express the protein
on one or more other
tissues, for example, in the muscle and/or brain. Such LSP-H, LSP-M and LSP-L
promoters disclosed
herein can preferentially express at least 90%, or at least 80%, or at least
70% or at least 60%, or at
least 50% of a protein in the liver, and also express at least 10%, or at
least 20%, or at least 30%, or at
least 40% or at least 50% in another tissue, for example, in muscle tissue. In
some embodiments, a
LSP-H, LSP-M and LSP-L promoter useful in the method and compositions as
disclosed herein, for
example for the treatment of Pompe or a lysosomal disease drives or enhances
gene expression in a
preferential or predominant manner in the liver, but can also express at least
some of the protein in
muscle tissue.
1001381The terms "identity" and "identical" and the like refer to the sequence
similarity between two
polymeric molecules, e.g., between two nucleic acid molecules, such as between
two DNA molecules.
Sequence alignments and determination of sequence identity can be done, e.g.,
using the Basic Local
Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J
Mol Biol 215: 403-
10), such as the "Blast 2 sequences" algorithm described by Tatusova and
Madden 1999 (FEMS
Microbiol Lett 174: 247-250).
1001391 The term "synthetic" as used herein means a nucleic acid molecule that
does not occur in
nature, Synthetic nucleic acid expression constructs of the present invention
are produced artificially,
typically by recombinant technologies. Such synthetic nucleic acids may
contain naturally occurring
sequences (e.g. promoter, enhancer, intron, and other such regulatory
sequences), but these are present
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in a non-naturally occurring context. For example, a synthetic gene (or
portion of a gene) typically
contains one or more nucleic acid sequences that are not contiguous in nature
(chimeric sequences),
and/or may encompass substitutions, insertions, and deletions and combinations
thereof.
[001401A "spacer sequence" or "spacer" as used herein is a nucleic acid
sequence that separates two
functional nucleic acid sequences. It can have essentially any sequence,
provided it does not prevent
the functional nucleic acid sequence (e.g. cis-regulatory element) from
functioning as desired (e.g.
this could happen if it includes a silencer sequence, prevents binding of the
desired transcription
factor, or suchlike). Typically, it is non-functional, as in it is present
only to space adjacent finictional
nucleic acid sequences from one another.
1001411The term "pharmaceutically acceptable" as used herein is consistent
with the art and means
compatible with the other ingredients of the pharmaceutical composition and
not deleterious to the
recipient thereof
[00142] By the terms "treat," "treating" or "treatment of' (and grammatical
variations thereof) it is
meant that the severity of the subject's condition is reduced, at least
partially improved or stabilized
and/or that some alleviation, mitigation, decrease or stabilization in at
least one clinical symptom is
achieved and/or there is a delay in the progression of the disease or
disorder.
1001431 The terms "prevent," "preventing" and "prevention" (and grammatical
variations thereof)
refer to prevention and/or delay of the onset of a disease, disorder and/or a
clinical symptom(s) in a
subject and/or a reduction in the severity of the onset of the disease,
disorder and/or clinical
symptom(s) relative to what would occur in the absence of the methods of the
invention. The
prevention can be complete, e.g., the total absence of the disease, disorder
and/or clinical symptom(s).
The prevention can also be partial, such that the occurrence of the disease,
disorder and/or clinical
symptom(s) in the subject and/or the severity of onset is substantially less
than what would occur in
the absence of the present invention.
[00144] A "treatment effective" amount as used herein is an amount that is
sufficient to provide
some improvement or benefit to the subject_ Alternatively stated, a "treatment
effective" amount is an
amount that will provide some alleviation, mitigation, decrease or
stabilization in at least one clinical
symptom in the subject. Those skilled in the art will appreciate that the
therapeutic effects need not
be complete or curative, as long as some benefit is provided to the subject.
[00145] A "prevention effective" amount as used herein is an amount that is
sufficient to prevent
and/or delay the onset of a disease, disorder and/or clinical symptoms in a
subject and/or to reduce
and/or delay the severity of the onset of a disease, disorder and/or clinical
symptoms in a subject
relative to what would occur in the absence of the methods of the invention.
Those skilled in the art
will appreciate that the level of prevention need not be complete, as long as
some preventative benefit
is provided to the subject.
[00146] The phrase a 'Therapeutically effective amount" and like phrases mean
a dose or plasma
concentration in a subject that provides the desired specific pharmacological
effect, e.g. to express a
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therapeutic gene in the liver, and secretion into the plasma. It is emphasized
that a therapeutically
effective amount may not always be effective in treating the conditions
described herein, even though
such dosage is deemed to be a therapeutically effective amount by those of
skill in the art. The
therapeutically effective amount may vary based on the route of administuation
and dosage form, the
age and weight of the subject, and/or the disease or condition being treated.
1001471 The terms "individual," "subject," and "patient" are used
interchangeably, and refer to any
individual subject with a disease or condition in need of treatment. For the
purposes of the present
disclosure, the subject may be a primate, preferably a human, or another
mammal, such as a dog, cat,
horse, pig, goat, or bovine, and the like.
[00148] Additional patents incorporated for reference herein that are related
to, disclose or describe
an AAV or an aspect of an AAV, including the DNA vector that includes the gene
of interest to be
expressed are: U.S. Patent Nos. 6,491,907; 7,229,823; 7,790,154; 7,201898;
7,071,172; 7,892,809;
7,867,484; 8,889,641; 9,169,494; 9,169,492; 9,441,206; 9,409,953; and,
9,447,433; 9,592,247; and,
9,737,618.
II. rAAV genome elements
[00149] As disclosed herein, one aspect of the technology relates to a rAAV
vector comprising a
capsid, and within its capsid, a nucleotide sequence referred to as the "rAAV
vector genome". The
rAAV vector genome (also referred to as "rAAV genome) includes multiple
elements, including, but
not limited to two inverted terminal repeats (ITRs, e.g., the 5'-ITR and the
3'-ITR), and located
between the ITRs are additional elements, including a promoter, a heterologous
gene and a poly-A
tail.
[00150] In some embodiments, the rAAV genome disclosed herein comprises a 5'
ITR and 3' ITR
sequence, and located between the 5'ITR and the 3' ITR, a promoter, e.g., a
liver specific promoter
sequence as disclosed herein, which operatively linked to a heterologous
nucleic acid encoding a
nucleic acid encoding an alpha-glucosidase (GAA) polypeptide, where the
heterologous nucleic acid
sequence can optionally further comprise one or more of the following
elements: an intron sequence,
a nucleic acid encoding a secretory signal peptide, a nucleic acid encoding an
IGF2 targeting peptide,
and a poly A sequence.
[00151] In some embodiments, the rAAV genome disclosed herein comprises a 5'
ITR and 3' ITR
sequence, and located between the 51TR and the 3' ITR, a promoter operatively
linked to a
heterologous nucleic acid encoding a secretory peptide and nucleic acid
encoding an alpha-
gluoosidase (GAA) polypeptide (i.e., the heterologous nucleic acid encodes a
GAA fusion polypeptide
comprising a signal peptide-GAA polypeptide), where the rAAV genome optionally
further comprises
one or more of: an intron sequence, a collagen stability (CS) sequence, a
polyA tail and a nucleic acid
encoding a spacer of at least 1 amino acid. In some embodiments, the rAAV
genome disclosed herein
comprises a 5' ITR and 3' ITR sequence, and located between the 5'1TR and the
3' ITR, a liver
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specific promoter as disclosed herein operatively linked to a heterologous
nucleic acid encoding a
secretory peptide (e.g., FN1, AAT or GAA signal peptides) and nucleic acid
encoding an alpha-
glucosidase (GAA) polypeptide, where the rAAV genome optionally further
comprises one or more
of: an intron sequence (e.g., MVM or 1-LBB2 intron sequence), a collagen
stability (CS) sequence, a
polyA tail and a nucleic acid encoding a spacer of at least 1 amino acid.
1001521 In some embodiments, the rAAV genome disclosed herein comprises a 5'
ITR and 3' ITR
sequence, and located between the 5111Z and the 3' ITR, a promoter operatively
linked to a
heterologous nucleic acid encoding a secretory peptide, a targeting peptide
and a GAA polypeptide
(i.e., the heterologous nucleic acid encodes a GAA fusion polypeptide
comprising a signal peptide-
targeting sequence-GAA polypeptide), where targeting peptide is a IGF2
targeting peptide as
described herein, and where the rAAV genome can optionally further comprise
one or more of: an
intron sequence, a collagen stability (CS) sequence, a polyA tail and a
nucleic acid encoding a spacer
of at least 1 amino acid.
1001531 Fait of the elements in the rAAV genome are discussed herein.
A. Alpha-glucosidase (GAA) polypeptide
1001541Alpha-gluposidase (GAA) polypeptide is a member of family 31 of
glycoside hydrolyases.
Human GAA is synthesized as a 110 kDal precursor (Wisselaar et al. (1993) J.
Biol. Chem. 268(3):
2223-31). The mature form of the enzyme is a mixture of monomers of 70 and 76
kDal (Wisselaar et
al. (1993) J. Biol. Chem. 268(3): 2223-31). The precursor enzyme has seven
potential glycosylation
sites and four of these are retained in the mature enzyme (Wisselaar et al.
(1993) J. Biol. Chem.
268(3): 2223-31). The proteolytic cleavage events which produce the mature
enzyme occur in late
endosomes or in the lysosome (Wisselaar et al. (1993) J. Biol. Chem. 268(3);
2223-31).
1001551 The rAAV vector genome can encode a GAA polypeptide can include, for
example, amino
acid residues 40-952 or 70-952 of human GAA, or a smaller portion, such as
amino acid residues 40-
790 or 70-790.
1001561 In one embodiment, the GAA polypeptide can be fused to an IGF2
targeting sequence. In
some embodiments, a IGF2 targeting sequence is fused to amino acid 40, or
amino acid 70, or to an
amino acid within one or two positions of amino acid 40 or 70 of human GAA
polypeptide. In some
embodiments, the IGF2 targeting peptide as disclosed herein is a ligand for an
extracellular receptor,
for example, the IGF2 targeting peptide binds to human cation-independent
mannose-6-phosphate
receptor (CI-MPR) or the IGF2 receptor.
1001571The C-terminal 160 amino acids are absent from the mature 70 and 76
kDal GAA polypeptide
species. However, certain Pompe alleles resulting in the complete loss of GAA
activity map to this
region, for example Va1949Asp (Becker et al. (1998) J. Hum, Genet 62:991). The
phenotype of this
mutant indicates that the C-terminal portion of the protein, although not part
of the 70 or 76 kDal
species, plays an important role in the function of the protein. It has also
been reported that the C-
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terminal portion of the protein, although cleaved from the rest of the protein
during processing,
remains associated with the major species (Moreland et al. (Nov. 1, 2004) J.
Biol. Chem., Manuscript
404008200). Accordingly, the C-terminal residues could play a direct role in
the catalytic activity of
the protein, and/or may be involved in promoting proper folding of the N-
tenninal portions of the
protein.
1001581The native GAA gene encodes a precursor polypeptide which possesses a
signal sequence and
an adjacent putative trans-membrane domain, a trefoil domain (PFAM PF00088)
which is a cysteine-
rich domain of about 45 amino acids containing 3 disulfide linkages (Thim
(1989) FEBS Lett.
250:85), the domain defined by the mature 70/76 kDal polypeptide, and the C-
terminal domain. It has
been reported that both the trefoil domain and the C-terminal domain are
required for the production
of finictional GAA, and that it is possible that the C-terminal domain
interacts with the trefoil domain
during protein folding perhaps facilitating appropriate disulfide bond
formation in the trefoil domain.
1001591The GAA polypeptide is described in US patents 5,962,313 and 6,537,785,
which are
incorporated herein in their entireties by reference. One of ordinary skill in
the art can appreciate
particular positions of GAA to which a secretory signal peptide (SS) or
alternatively, the targeting
peptide (e.g., IGF2 targeting peptide) can be fused. Accordingly, in one
aspect the invention relates to
a GAA fusion protein, where the SP or IGF2 targeting peptide is fused to amino
acid 40, 68, 69, 70,
71, 72, 779, 787, 789, 790, 791, 792, 793, or 796 of human GAA of SEQ ID NO:
10, or a modified
GAA protein of SEQ ID NO: 170-174, or a portion thereof.
1001601ln some embodiments of the methods and compositions as disclosed
herein, the human GAA
protein expressed by the AAV comprises amino acids of SEQ ID NO: 10, or
fragments or variants
thereof, for example a human GAA protein beginning at residue 40, 68, 69, 70,
71, 72, 779, 787, 789,
790, 791, 792, 793, or 796 of SEQ ID NO: 10. In some embodiments of the
methods and
compositions as disclosed herein, the human GAA protein expressed by the AAV
comprises amino
acids of SEQ ID NO: 10, or a protein at least 60%, or 70%, or 80%, 85% or 90%
or 95%, or 98%, or
99% identical to SEQ ID NO: 10. In some embodiments of the methods and
compositions as
disclosed herein, the human GAA protein expressed by the AAV comprises amino
acids is a human
GAA protein beginning at residue 40, 68, 69, 70, 71, 72, 779, 787, 789, 790,
791, 792, 793, or 796 of
SEQ ID NO: 10, or a protein at least 60%, or 70%, or 80%, 85% or 90% or 95%,
or 98%, or 99%
identical thereto. In some embodiments, the human GAA protein expressed by the
AAV comprises
amino acids of beginning at residue 40, 68, 69, 70, 71, 72, 779, 787, 789,
790, 791, 792, 793, or 796
of any of SEQ ID NO: 170 (modGAA; H199R, R223H) or SEQ ID NO: 171 (modGAA;
H199R,
R223H, H201L) or a protein at least 60%, or 70%, or 80%, 85% or 90% or 95%, or
98%, or 99%
identical thereto.
1001611 In some embodiments, one of ordinary skill in the art can appreciate
particular positions of
GAA to which a secretory signal peptide (SS) or alternatively, the targeting
peptide (e.g., IGF2
targeting peptide) can be fused. For example, International Patent application
W02018046774A1,
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which is incorporated herein in its entirety, discloses truncated GAA
polypeptides to which the
secretory signal peptide (SS) or alternatively, the targeting peptide (e.g.,
IGF2 targeting peptide) can
be attached. The signal peptide or IGF2 targeting peptide can be attached to
any truncated GAA
polypeptide or truncated modified GAA polypeptide, starting amino acids of GAA
truncated proteins
as disclosed in U.S. Provisional Application 62,937,556, filed on November 19,
2019 and
International Application WO 2020/102667, filed Nov 15, 2019. which is
incorporated herein in its
entirety by reference.
1001621In some embodiments, the GAA-fusion polypeptides encoded by the rAAV
genome as
described herein can include, for example, amino acid residues 40-952 or
residues 70-952 of human
GAA, or a smaller portion, such as amino acid residues 40-790 or 70-790. In
one embodiment, a
secretory signal peptide (SS) or targeting peptide, e.g., IGF2 targeting
peptide is fused to amino acid
40, or to amino acid 70, or to an amino acid within one or two positions of
amino acid 40 or 70.
1001631 In some embodiments, the fusion protein comprising the secretory
signal peptide (SS) and
GAA polypeptide and optionally an IGF2 targeting peptide (i.e., a SS-GAA
fusion polypeptide, or a
SS-IGF2-GAA fusion protein) comprises amino acid residues 40-952 or residues
70-952 of human
acid alpha-glucosidase (GAA) (SEQ ID NO: 10). In some embodiments, the N-
terminal of the GAA
polypeptide is attached to the C-tenuinus of the SS and in some embodiments,
the N-tenninal of the
GAA polypeptide is attached to the C-terminus of the IGF2 targeting peptide,
and the N-terminus of
the IGF2 targeting peptide is attached to the C-terminus of the secretory
signal peptide.
Modified GAA (modGAA)
1001641 In some embodiments, the GAA protein comprises a H201L variant, as
disclosed in
US2014/0186326, and Moreland et at, Gene, 2012; 491 (25-30), which are both
incorporated herein
in their entirety by reference. In particular, the histidine (His) at amino
acid position 201 is changed to
a leucine (L) residue to enables rapid processing of the 76kD GAA pre-protein
into the mature 70kD
GAA protein.
10016511n particular, in some embodiments, a fusion protein as disclosed
herein comprises a GAA
polypeptide of SEQ ID NO: 10, with a modification of amino acids that results
in increased
hydrophobicity at or near the N-terminal 70-kDa processing site. In some
instances, the GAA peptide
is modified at one or more amino acids corresponding to positions 190-209 of
SEQ ID NO: 10. In
further embodiments, the polypeptide is modified at one or more amino acids
corresponding to
positions 195-209 of SEQ ID NO: 10. In further embodiments, the modification
is at one or more
amino acids corresponding to amino acid positions 200-204 of SEQ ID NO: 10. In
certain
embodiments, the modification is at the amino acid corresponding to position
201 of SEQ ID NO: 10.
In further embodiments, the modification is substitution of one or more amino
acids with a more
hydrophobic amino acid. In other embodiments, the modification is insertion of
one or more
hydrophobic amino acids. In even further embodiments, the hydrophobic amino
acid is chosen from
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leucine and tyrosine, or a conservative amino acid of leucine or tyrosine.
1001661In certain embodiments, GAA is modified to increase its hydrophobicity
at or near the N-
terminal 70-kDa processing site by substituting at least one amino acid with a
more hydrophobic
amino acid. In some embodiments, the substitution may be made within 5 amino
acids upstream or
downstream of the N-terminal 70-kDa processing site. In certain examples, the
amino acid
substitution may be made at an amino acid corresponding to position 195 to 209
of SEQ ID NO: 10.
In other instances, the amino acid substitution may be made at an amino acid
corresponding to
position 200 to 204 of SEQ ID NO: 10. In further embodiments, the modified
human GAA contains a
hydrophobic amino acid at the position corresponding to amino acid position
201 of SEQ ID NO: 10.
In some embodiments, GAA is modified by inserting one or more hydrophobic
amino acids at or near
the N-terminal 70-kDa processing site. Additional modifications include
deletion of one or more
amino acids at or near the N-terminal 70-kDa processing site.
1001671In certain embodiments, a modified human GAA is provided containing a
hydrophobic amino
acid (natural or synthetic) at more than one position at the N-terminal 70-kDa
processing site, or
within 5 amino acids of the N-terminal 70-kDa processing site. In one
embodiment, one of the
modified amino acids is at the position corresponding to amino acid 201 of SEQ
ID NO: 10,
1001681ln various embodiments the hydrophobic amino acid is chosen from
valine, leucine,
isoleucine, methionine, phenylalanine, tryptophan, tyrosine, cysteine or
alanine. In further
embodiments, the hydrophobic amino acid is leucine or tyrosine. In some
embodiments, the modified
human GAA contains a synthetic or non-natural amino acid that exhibits
hydrophobic properties.
Generally, the substituted amino acid is more hydrophobic than the wild-type
amino acid, and thus
increases the hydrophobicity at or near the N-terminal 70kDa processing site.
1001691In one exemplary embodiment, the modified GAA has a leucine at the
position corresponding
to amino acid 201 of SEQ ID NO: 10. In another embodiment, the modified GAA
has a tyrosine at the
position corresponding to amino acid 201 of SEQ ID NO: 10.
1001701In some embodiments, the modified human GAA protein comprises a
polypeptide with a His
(H) to Arginine (R) (H199R) modification at amino acid position 199 of SEQ ID
NO: 10
(GAA(H199R), or a modification of an arginine (R) to a histidine (H) (R223H)
at amino acid position
223 of SEQ ID NO: 10 (GAA(R223H). In some embodiments, the modified human GAA
protein
comprises a polypeptide with a His (H) to Arginine (R) (Hi 99R) modification
at amino acid position
199 of SEQ ID NO: 10, and a modification of an arginine (R) to a histidine (H)
(R223H) at amino
acid position 223 of SEQ ID NO: 10 (GAA(H199R-R223H). In some embodiments, the
modified
human GAA protein comprises SE() ID NO: 170 or a variant of at least 80%, 90%,
95%, or 99%
homology to at least 500, 550, 600, 650, 700, 750, 800, 850, or 900 amino
acids of SEQ ID NO: 170,
having at least one modification of H199R or R223H, or both. In some
embodiments, the cognate
leader sequence of GAA (i.e., SEQ ID NO: 175 or amino acids 1-27 of SEQ ID NO:
170) is replaced
with an IGF2 targeting peptide as disclosed herein, or a leader sequence of
SEQ ID NO: 176, or an
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IL2 wild type leader peptide (SEQ ID NO: 178), modified IL2 leader peptide
(SEQ ID NO: 180) or
leader peptides at least 90% sequence identity to SEQ ID Nos 176, 178 or 180.
[00171] In some embodiments, the modified human GAA protein comprises SEQ ID
NO: 171 or a
variant of at least 80%, 90%, 95%, 01 99% homology to at least 500, 550, 600,
650, 700, 750, 800,
850, or 900 amino acids of SEQ ID NO: 171, comprising at least the
modification of H210L. In some
embodiments, the cognate leader sequence of GAA (i.e., SEQ ID NO: 175 or amino
acids 1-27 of
SEQ ID NO: 171) is replaced with an IGF2 targeting peptide as disclosed
herein, or a leader sequence
of SEQ ID NO: 176, or an IL2 wild type leader peptide (SEQ ID NO: 178),
modified IL2 leader
peptide (SEQ ID NO: 180) or leader peptides at least 90% sequence identity to
SEQ ID Nos 176, 178
or 180
1001721 In some embodiments, the modified human GAA protein comprises a
polypeptide with at
least one modification selected from: H199R, R223H, or H201L of SEQ ID NO: 10,
or a variant of at
least 80%, 90%, 95%, or 99% homology to at least 500, 550, 600, 650, 700, 750,
800, 850, or 900
amino acids of SEQ ID NO: 10 having at least one of these modification. In
some embodiments, the
modified human GAA protein comprises a polypeptide comprises at least two
modifications selected
from: H199R, R223H, or H201L of SEQ ID NO: 10, or a variant of at least 80%,
90%, 95%, or 99%
homology to at least 500, 550, 600, 650, 700, 750, 800, 850, or 900 amino
acids of SEQ ID NO: 10
having at least two of these modification. In some embodiments, the modified
human GAA protein
comprises a polypeptide with three modifications Hi 99R, R223H, and H201L of
SEQ ID NO: 10
((fAA- H199R-H201L- R223H), or a variant of at least 80%, 90%, 95%, or 99%
homology to at least
500, 550, 600, 650, 700, 750, 800, 850, or 900 amino acids of SEQ ID NO: 10
having these three
modifications.
1001731 In certain embodiments, modified human GAAs are provided having at
least 80%, 90%, 95%,
or 99% homology to at least 500, 550, 600, 650, 700, 750, 800, 850, or 900
amino acids of SEQ ID
NO: 10, and wherein the modified human GAA has at least one amino acid in the
N-terminal 70-kDa
processing site substituted with a more hydrophobic amino acid.
10017411n some embodiments, at least 50% of the modified human GAA is
processed to a 70-kDa
form in the lysosome within 20, 30, or 40 hours. In still further embodiments,
substantially all of the
modified human GAA is processed to a 70-kDa form in the lysosome within 55,
65, or 75 hours.
1001751ln certain embodiments, a modified human GAA of the invention can be
identified by its
more rapid proteolytic processing to a mature 70-kDa form, or a corresponding
variant thereof. In
other embodiments, a modified human GAA as described herein can be identified
by the production
of an 82-kDa intermediate polypeptide that is not produced during proteolytic
processing of native
human GAA. In further embodiments, a modified human GAA can be identified by
the absence of a
76-kna intermediate polypeptide that is produced during proteolytic processing
of unmodified human
GAA.
1001761ln certain embodiments, the polypeptide has at least 80% identity to at
least 500 amino acids
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of SEQ ID NO: 10 or SEQ ID NO: 170-171. In some instances, the polypeptide has
at least 90%
identity to at least 500 amino acids of SEQ ID NO: 10 or SEQ ID NO: 170-171.
In other instances,
the polypeptide has at least 95% identity to at least 500 amino acids of SEQ
ID NO: 10 or SEQ ID
NO: 170-171.
1001771In certain embodiments, the GAA polypeptide with a modification at
amino acid 201 to a
hydrophobic residue, e.g., for example a H201L modification, exhibits more
rapid lysosomal protease
processing when compared to an unmodified human acid alpha-glucosidase
protein. In some
embodiments, at least 50% of the GAA pre-polypeptide is proteolytically
processed to a 70-kDa
mature GAA form within 20 hours of expression. In other embodiments,
substantially all the GAA
pre-polypeptide is proteolytically processed to a 70-kDa mature GAA form
within 55 hours of
expression.
1001781ln some embodiments, the cognate GAA leader peptide of amino acids 1-27
of SEQ ID NO:
(i.e., MGVRHPPCSHRLLAVCALVSLATAALL, SEQ ID NO: 175) is replaced with a
different
signal peptide (leader peptide). For example, the cognate leader peptide of
GAA (SEQ ID NO: 175)
can be replaced with any of: (i) an 1861 leader peptide (referred to herein as
a "201 leader peptide" or
"2011p" having an amino acid sequence of: MEFGLSWVFLVALLKGVQCE (SEQ ID NO:
176)
encoded by nucleic acid sequence SEQ ID NO: 177, (ii) wtIL2 1p:
MYRMQLLSCIALSLALVTNS
(SEQ ID NO: 178) encoded by nucleic acid sequence SEQ ID NO: 179, or (iii)
mutIL2 1p:
MYRMQLLLL/ALSLALVTNS (SEQ ID NO: 180) encoded by nucleic acid sequence SEQ ID
NO:
181. In some embodiments, the cognate GAA leader peptide (SEQ ID NO: 175)
remains present, and
an additional signal peptide is added, e.g., any one or more of signal
peptides AAT, FN1, an IgG1
leader peptide (referred to herein as a "201 leader peptide" or "2011p" having
an amino acid sequence
of: MEFGLSWVFLVALLKGVQCE (SEQ ID NO: 176) encoded by nucleic acid sequence SEQ
ID
NO: 177, (ii) w-tIL2 1p: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 178) encoded by
nucleic acid
sequence SEQ ID NO: 179, or (iii) mutIL2 1p: MYRIV1QLLLL/ALSLALVTNS (SEQ ID
NO: 180)
encoded by nucleic acid sequence SEQ ID NO: 181
1001791ln some embodiments, GAA is modified to add or remove glycosylation
sites such as N-
linked glycosylation sites, 04inked glycosylation sites or both. In certain
embodiments, the addition
or removal of glycosylation sites are achieved by N-terminal deletions, C-
terminal deletions, internal
deletions, random point mutagenesis, or, site directed mutagenesis. In some
embodiments, the
exemplary GAA modification involve addition of one or more Asparagine (Asn)
residue/s or, one or
more mutation to yield Asparagine (Asn) residue/s or, deletion of one or more
Asparagine (Asn)
residue/s. In certain embodiments, all or some of the N-linked, and/or, 0-
linked glycosylation sites
present in GAA are mutated. In some embodiments, GAA modifications will yield
information
pertaining to the biological activity, physical structure and/or substrate
binding potential of GAA.
(ii) Nucleic acid encoding GAA
1001801 In some embodiments, the rAAV genome comprises a heterologous nucleic
acid sequence
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encoding the entire GAA polypeptide (e.g., the N-terminalkatalytic and the C-
terminal domain), that
is not fused to a heterologous signal sequence or a targeting peptide.
[00181] In some embodiments, the rAAV genome comprises a heterologous nucleic
acid sequence
encoding a secretory signal peptide or IGF2 targeting peptide fused in frame
to the 3' terminus of a
GAA nucleic acid sequence that encodes the entire GAA polypeptide (e.g., the N-
terminalkatalytic
and the C-terminal domain). For example, heterologous nucleic acid sequence
encoding a secretory
signal peptide, or IGF2 targeting peptide is fused in frame to the 3' terminus
of a GAA nucleic acid
sequence that encodes the 70kDa and 76 kDa GAA polypeptides, such both
polypeptides are
expressed from the rAAV genome when the rAAV vector transduces a mammalian
cell. In some
embodiments, expression of the GAA nucleic acid can be driven by two promoters
in the rAAV
genome or by one promoter driving expression of a bicistronic construct.
[00182] In some embodiments of the methods and compositions as disclosed
herein, the rAAV
vector comprises a nucleic acid sequence encoding a GAA protein is a wild type
GAA nucleic acid
sequence, e.g., SEQ ID NO: 11 or SEQ ID NO: 72 or SEQ ID NO: 182. In some
embodiments of the
methods and compositions as disclosed herein, the rAAV vector comprises a
nucleic acid sequence
encoding a GAA protein which is a codon optimized GAA nucleic acid sequence,
for any one or more
of (i) enhanced expression in vivo, (ii) to reduce CpG islands, (iii).to
reduce the innate immune
response. Exemplary codon optimized GAA nucleic sequences encompassed for use
in the methods
and rAAV compositions as disclosed herein can be selected from any of: SEQ ID
NO: 73, SEQ ID
NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 182, or a nucleic acid
sequence having at
least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity
to SEQ ID NO:
73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 182.
[00183] In addition, in some embodiments, the GAA nucleic acid sequences
encompassed for use in
the methods and rAAV compositions as disclosed herein are further modified
with at least one or
more of the following modifications: (i) removal of at least one, or two or in
some embodiments, all
alternative reading frames, (ii) removal of one or more CpGs islands, (iii)
modification of the Kozak
sequence, (iv) modification of a translational terminator sequence, and (v)
removal of a spacer
between promoter and Kozak sequence.
[00184] For example, in some embodiments, the rAAV composition comprises a
hGAA nucleotide
sequence of SEQ ID NO: 182, or a nucleic acid sequence having at least 60%, or
70%, or 80%, 85%
or 90% or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 182, where SEQ
ID NO: 182
comprises the following elements shown in Table 1A, as compared to the wild
type nucleic acid
sequence for GAA;
[00185] Table 1A: elements of modGAA nucleic acid sequence.
modification Base
pair (nucleotide numbers
based on SEQ ID NO: 440)
cognate leader peptide 954-
1034 (8 lbp)
GAT to GAC for Asp (T154C) 1152-
1154
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AST to AAC for Asn (T1901C) 1899-1901
GAT to GAC for Asp (T1910C) 1909-1910
GAT to GAC for Asp (T1967C) 1965-1967
GTT to GTG to remove CpG 2022-
2024
(T2024G)
CAA to CAG for gin (A2156G) 2154-2156
CAC to GAT to remove CpG 2163-
2165
(C2165T)
GTA to GTG for Val (A2393G) 2391-2393
AAT to AAC for Asn (T2561C) 2259-2561
GTC to GTG to remove CpG 3399-
3401
(C3401G)
GAT to GAC for Asp (T3536C) 3534-3536
Replacement of non optimal 3810-
3821 (12bp)
stop
[00186] In some in some embodiments, the rAAV composition comprises a hGAA
nucleotide
sequence of SEQ ID NO: 182, or a nucleic acid sequence having at least 60%, or
70%, or 80%, 85%
or 90% or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 182, where the
hGAA nucleotide
sequence has been modified to with a series of point mutations that eliminate
3 potentially pro-
inflammatory CpG motifs and a number of alternative reading frames (ARFs),
where SEQ ID NO:
182 comprises the following point mutations as shown in Table 1B, as compared
to the wildtype
nucleic acid sequence for GAA, where numbering in table 1B assumes "A" in the
GAA start codon
ATG is the first nucleotide.
[00187] Table 1B:
NT# Purpose Original NT New NT NT# Purpose
Original New NT
NT
201 Remove ORF T C
1212 Destroy CpG
949 Remove ORF T C
1440 Remove ORF A
957 Remove ORF T C
1608 Remove ORF
1014 Remove ORF T C
2448 Destroy CpG
1071 Destroy CpG T U
2583 Remove ORF
1203 Remove ORF A
[00188] In some embodiments, the nucleic acid sequence encoding the cognate
leader peptide in SEQ
ID NO: 182 (e.g., nucleotides 1-81 of SEQ ID NO: 182) can be replaced by
nucleic acid sequences
encoding any of 201lp, wtIL2 1p or mutIL2 1p. Accordingly, in some
embodiments, the nucleic
residues 1-81 of SEQ ID NO: 182 (encoding the cognate leader peptide of GAA)
can be replaced by
nucleic acid sequences of SEQ ID NO: 177 (2011p), SEQ ID NO: 179 (wtIL21p) or
SEQ ID NO: 181
(mutIL2 1p), or a nucleic acid sequence having at least 60%, or 70%, or 80%,
85% or 90% or 95%, or
98%, or 99% sequence identity to SEQ ID NOS: 177, 179 or 181.
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[0018911n some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence encoding a GAA polypeptide comprising SEQ ID NO: 170 (GAA
polypeptide with a
cognate GAA signal sequence and H199R, R223H modifications), or SEQ ID NO; 171
(GAA
polypeptide with a cognate GAA signal sequence and H199Rõ H201L, R223H
modifications). The
GAA polypeptide of SEQ ID NO: 170 is encoded by the nucleic acid sequence of
SEQ ID NO: 182.
Accordingly, in some embodiments, the rAAV vector comprises a nucleic acid of
SEQ ID NO: 182
encoding a modified GAA polypeptide comprising H199R, R223H modifications. The
GAA
polypeptide of SEQ ID NO: 171 is encoded by the nucleic acid sequence of SEQ
ID NO: 182 where
basepairs (bp) 667-669 of SEQ ID NO: 182 are changed from CAC to any of: UUA,
UUG, CUU,
CUC CUA, CUG (resulting in a Histadine (H) to Leucine (L) amino acid change);
or where bp 668 of
SEQ ID NO: 182 is changed from A to U. Accordingly, in some embodiments, the
rAAV vector
comprises a nucleic acid of SEQ ID NO: 182, where bp 667-669 of SEQ ID NO: 182
are changed
from CAC to any of UUA, UUG, CUU, CUC CUA, CUG (which changes the amino acid
from
Histidine (H) to leucine (L)); or where bp 668 of SEQ ID NO: 182 is changed
from A to U, which
encodes a modified GAA polypeptide comprising H199R, H201L and R223H
modifications.
[00190] In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence encoding a GAA polypeptide selected from any of: SEQ ID NO: 172
(GAA
polypeptide where cognate signal peptide is replaced with a IgG signal
sequence and H199R, R223H
modifications), or SEQ ID NO: 173 (GAA polypeptide where cognate signal
peptide is replaced with
a wtIL2 signal sequence and H199R, R223H modifications), SEQ ID NO: 174 ((fAA
polypeptide
where cognate signal peptide is replaced with a mutIL3 signal sequence and
H199R, R223H
modifications).
[0019111n some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 177 (IgG signal sequence), which encodes a GAA
polypeptide of SEQ
ID NO: 172 (IgG leader-GAA with H199R, R223H modifications). In some
embodiments, the rAAV
vector comprises a heterologous nucleic acid sequence comprising SEQ ID NO:
182, where bp 668 of
SEQ ID NO: 182 is changed from A to U and where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 177 (IgG signal peptide), which encodes a GAA
polypeptide of SEQ ID
NO: 172 (1gG leader-GAA with H199R, H201L and 11223H modifications).
1001921 In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 179 (wt IL2 signal peptide), which encodes a GAA
polypeptide of SEQ
ID NO: 173 (wt IL2 signal peptide-GAA with H199R, R223H modifications). In
some embodiments,
the rAAV vector comprises a heterologous nucleic acid sequence comprising SEQ
ID NO: 182, where
bp 668 of SEQ ID NO: 182 is changed from A to U and where bp 1-81 of SEQ ID
NO: 182 is
replaced with the nucleic acid of SEQ ID NO: 179 (wt IL2 signal peptide),
which encodes a GAA
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polypeptide of SEQ ID NO: 173 (wt IL2 signal peptide-GAA with H199R, H201L and
R223H
modifications).
[00193] In some embodiments, the rAAV vector comprises a heterologous nucleic
acid sequence
comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is replaced with the
nucleic acid of
SEQ ID NO: 181 (mutIL2 signal peptide), which encodes a GAA polypeptide of SEQ
ID NO: 174
(mutIL2 signal peptide-GAA with H199R, R223H modifications). In some
embodiments, the rAAV
vector comprises a heterologous nucleic acid sequence comprising SEQ ID NO:
182, where bp 668 of
SEQ ID NO: 182 is changed from A to U and where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 181 (mut IL2 signal peptide), which encodes a GAA
polypeptide of SEQ
ID NO: 174 (mut IL2 signal peptide-GAA with H199R, H201L, and R223H
modifications).
[00194] The C-terminal domain of GAA functions in trans in conjunction with
the 70/76 kDal species
to generate active GAA. The boundary between the catalytic domain and the C-
terminal domain
appears to be at about amino acid residue 791, based on its presence in a
short region of less than 18
amino acids that is absent from most members of the family 31 hydrolyases and
which contains 4
consecutive proline residues in GAA. It has been reported that the C-terminal
domain associated with
the mature species begins at amino acid residue 792 (Moreland et al. (Nov. 1,
2004)J Biol. Chew,
Manuscript 404008200). Accordingly, in some embodiments, the GAA nucleic acid
sequence that
encodes the entire GAA polypeptide, with the exception of the C-terminal
domain. Thus, in such an
embodiment, the rAAV vector can be used to transduce a mammalian cell that
expresses the C-
terminal domain of GAA as a separate polypeptide.
B. Secretory Signal peptide
[00195] Native GAA signal peptide is not cleaved in the ER thereby causing
native GAA
polypeptide to be membrane bound in the ER (Tsuji et al. (1987) Biochem. Int.
15(5):945-952).
Disruption of the membrane association of GAA can be accomplished by replacing
the endogenous
GAA signal peptide (and optionally adjacent sequences) with an alternate
signal peptide for GAA.
[00196] Accordingly, in representative embodiments, the rAAV vector and rAAV
genome as
disclosed herein further comprises a heterologous nucleic acid encoding a GAA
polypeptide to be
transferred to a target cell, attached to a heterologous nucleic acid sequence
that encodes a secretory
signal peptide in the place of the endogenous GAA signal peptide. The
heterologous nucleic acid is
operatively associated with the segment encoding the secretory signal peptide,
such that upon
transcription and translation a fusion polypeptide is produced containing the
secretory signal sequence
operably associated with (e.g., directing the secretion of) the GAA
polypeptide.
[00197] In some embodiments, the AAV vector encodes a GAA polypeptide that
comprises the
endogenous GAA signal peptide (e.g., amino acids 1-27 of SEQ ID NO: 10 (also
referred to as
"innate GAA" or "cognate (iAA" signal peptide). In some embodiments, the AAV
vector encodes a
GAA polypeptide that comprises the endogenous GAA signal peptide (e.g., amino
acids 1-27 of SEQ
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ID NO: 10 (also referred to as "innate GAA" or "cognate (IAA" signal peptide)
and an additional
heterologous (non native) signal sequence. In some embodiments, the GAA
polypeptide that lacks
the endogenous signal peptide of amino acids 1-27 of GAA is fused to a
secretory signal .In some
embodiments of the compositions and methods described herein, the secretory
signal serves a general
purpose of assisting the secretion of the GAA polypeptide, or a fusion
polypeptide, e.g., the IGF2
targeting peptide-GAA fusion polypeptide from the liver cells into the blood,
where it can travel and
be targeted to the lysosomes of mammalian cells, for example, human cardiac
and skeletal muscle
cells, as described herein. In some embodiments, a heterologous secretory
signal is selected from any
of: a AAT signal peptide, a fibronectin signal peptide (FN1), a GAA signal
peptide, or an active
fragment of AAT, FN1 or GAA signal peptide having secretory signal activity.
[00198] In some embodiments, the secretory signal peptide is heterologous to
(i.e., foreign or
exogenous to) the polypeptide of interest. For example, a heterologous
secretory signal peptide is a
fibronectin secretory signal peptide, the polypeptide of interest is not
fibronectin. In some
embodiments, the secretory signal peptide is selected from any of: AAT signal
peptide, a fibronectin
signal peptide (FN1), or an active fragment of AAT, FN1 or GAA signal peptide
having secretory
signal activity. In alternative embodiments, the secretory signal peptide is
not heterologous to GAA,
i.e., the signal peptide is The GAA signal peptide (i.e., residues 1-27 of the
native GAA polypeptide).
[00199] In some embodiments, the cognate GAA signal peptide of amino acids 1-
27 of SEQ ID NO:
(i.e., MGVRIIPPCSHRLLAVCALVSLATAALL, SEQ ID NO: 175) is replaced with a
different
or heterologous leader peptide. For example, the cognate leader peptide of GAA
(SEQ ID NO: 175)
can be replaced with any of the heterologous signal peptides selected from:
(i) an IgG1 leader peptide
(referred to herein as a "201 leader peptide" or "2011p" having an amino acid
sequence of:
MEFGLSWVFLVALLKGVQCE (SEQ ID NO: 176) encoded by nucleic acid sequence SEQ ID
NO:
177, (ii) wtIL2 1p: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 178) encoded by nucleic
acid
sequence SEQ ID NO: 179, or (iii) mutIL2 1p: MYRMQLLLL/ALSLALVTNS (SEQ ID NO:
180)
encoded by nucleic acid sequence SEQ ID NO: 181, or a leader peptide having at
least 90% sequence
identity to any of SEQ ID NOs 176, 178 or 180.
[00200] In general, the secretory signal peptide will be at the amino-terminus
(N-terminus) of the
fusion polypeptide (i.e., the nucleic acid segment encoding the secretory
signal peptide is 5 to the
heterologous nucleic acid encoding the GAA peptide or GAA-fusion peptide in
the rAAV vector or
rAAV genome as disclosed herein). Alternatively, the secretory signal may be
at the carboxyl-
terminus or embedded within the GAA polypeptide or GAA fusion polypeptide
(es., IGF2-GAA
fusion polypeptide), as long as the secretory signal is operatively associated
therewith and directs
secretion of the GAA polypeptide or GAA fusion polypeptide of interest (either
with or without
cleavage of the signal peptide from the GAA polypeptide) from the cell.
[00201] The secretory signal is operatively associated with the GAA
polypeptide or GAA fusion
polypeptide is targeted to the secretory pathway. Alternatively stated, the
secretory signal is
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operatively associated with the GAA polypeptide such that the GAA-polypeptide
or GAA fusion
polypeptide is secreted from the cell at a higher level (i.e., a greater
quantity) than in the absence of
the secretory signal peptide. In general, typically at least about 20%, 30%,
40%, 50%, 70%, 80%,
85%, 90%, 95% or more of the GAA-polypeptide or IGF2-GAA fusion polypeptide
(alone and/or
fused with the signal peptide) is secreted from the cell when a signal peptide
is attached as compared
to in the absence of the attachment of a secretory signal peptide. In other
embodiments, essentially all
of the detectable polypeptide (alone and/or in the form of the fusion
polypeptide) is secreted from the
cell.
1002021By the phrase "secreted from the cell", the polypeptide may be secreted
into any compartment
(e.g., fluid or space) outside of the cell including but not limited to: the
interstitial space, blood,
lymph, cerebrospinal fluid, kidney tubules, airway passages (e.g., alveoli,
bronchioles, bronchia, nasal
passages, etc.), the gastrointestinal tract (e.g., esophagus, stomach, small
intestine, colon, etc.),
vitreous fluid in the eye, and the cochlear endolymph, and the like.
1002031 In one embodiment, the rAAV genome comprises a heterologous nucleic
acid that encodes
a secretory signal peptide (SP) fused to the GAA-fusion polypeptide, where the
GAA-fusion
polypeptide comprises a targeting peptide (e.g., IGF2 targeting peptide) fused
to a GAA polypeptide.
As used herein GAA also refers to the modified GAA described above.
Accordingly, the signal
peptide disclosed herein increases the efficacy of secretion of the GAA
polypeptide or IGF2-GAA
fusion polypeptide from the cell transduced with the rAAV vector or comprising
the rAAV genome as
described herein
1002041 Accordingly, in some embodiments, the rAAV genome disclosed herein
comprises a 5' ITR
and 3' ITR sequence, and located between the 5'ITR and the 3' ITR, a promoter
operatively linked to
a heterologous nucleic acid encoding a secretory peptide and nucleic acid
encoding an alpha-
glucosidase (GAA) polypeptide (i.e., the heterologous nucleic acid encodes a
GAA fusion polypeptide
comprising a signal peptide-GAA polypeptide).
1002051 In alternative embodiments, the rAAV genome disclosed herein comprises
a 5' ITR and 3'
ITR sequence, and located between the 51ITR and the 3' FUR, a promoter
operatively linked to a
heterologous nucleic acid encoding a secretory peptide and nucleic acid
encoding an alpha-
glucosidase (GAA) fusion polypeptide, where the fusion protein comprises IGF2
targeting peptide
and a GAA polypeptide (i.e., the heterologous nucleic acid encodes a GAA
fusion polypeptide
comprising a signal peptide-IGF2-GAA polypeptide).
1002061 Generally, secretory signal peptides are cleaved within the
endoplasmic reticulum and, in
some embodiments, the secretory signal peptide is cleaved from the GAA
polypeptide prior to
secretion. It is not necessary, however, that the secretory signal peptide is
cleaved as long as secretion
of the GAA polypeptide or IGF2-GAA fusion polypeptide from the cell is
enhanced and the GAA
polypeptide is functional. Thus, in some embodiments, the secretory signal
peptide is partially or
entirely retained.
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1002071 In some embodiments, the rAAV genome, or an isolated nucleic acid as
disclosed herein
comprises a nucleic acid encoding a chimeric polypeptide comprising a GAA
polypeptide operably
linked to a secretory signal peptide, and the chimeric polypeptide is
expressed and produced from a
cell trartsduced with the rAAV vector and the GAA polypeptide is secreted from
the cell. The GAA
polypeptide or GAA fusion polypeptide (e.g., IGF2-GAA fusion polypeptide) can
be secreted after
cleavage of all or part of the secretory signal peptide. Alternatively, the
GAA polypeptide or GAA
fusion polypeptide (e.g., IGF2-GAA fusion polypeptide) can retain the
secretory signal peptide (i.e.,
the secretory signal is not cleaved). Thus, in this context, the "GAA
polypeptide or GAA fusion
polypeptide" can be a chimeric polypeptide comprising the secretory peptide.
1002081The secretory signal sequences of the invention are not limited to any
particular length as long
as they direct the polypeptide of interest to the secretory pathway. In
representative embodiments, the
signal peptide is at least about 6, 8, 10 12, 15, 20, 25, 30 or 35 amino acids
in length up to a length of
about 40, 50, 60, 75, or 100 amino acids or longer.
1002091 Secretory signal peptide encoded by the rAAV genome and in the rAAV
vector as disclosed
herein can comprise, consist essentially of or consist of a naturally
occurring secretory signal
sequence or a modification thereof. Numerous secreted proteins and sequences
that direct secretion
from the cell are known in the art, are disclosed in US Patent 9,873,868,
which is incorporated herein
in its entirety by reference. Exemplary secreted proteins (and their secretory
signals) include but are
not limited to: erythropoietin, coagulation Factor IX, cystatin,
lactotransferrin, plasma protease Cl
inhibitor, apolipoproteins (e.g., APO A, C, E), MCP-1, a-2-HS-glycoprotein, a-
l-microgolubilin,
complement (e.g., ClQ, C3), vitronectin, lymphotoxin-a, azurocidin, VIP,
metalloproteinase inhibitor
2, glypican-1, pancreatic hormone, clusterin, hepatocyte growth factor,
insulin, a-l-antichymotrypsin,
growth hormone, type IV collagenase, guanylin, properdin, proenkephalin A,
inhibin ft (e.g., A chain),
prealbumin, angiocenin, lutropin (e.g., p chain), insulin-like growth factor
binding protein 1 and 2,
proactivator polypeptide, fibrinogen (e.g., 13 chain), gastric triacylglycerol
lipase, midkine, neutrophil
defensins 1, 2, and 3, a- 1-antitrypsin, matrix gla-protein, a-tryptase, bile-
salt-activated lipase,
chymotrypsinogen B, elastin, IG lambda chain V region, platelet factor 4
variant, chromogranin A,
WNT-1 proto-oncogene protein, oncostatin M,13-neoendorphin-dynorphin, von
Willebrand factor,
plasma serine protease inhibitor, serum amyloid A protein, nidogen,
fibronectin, rennin, osteonectin,
histatin 3, phospholipase A2, cartilage matrix Protein, GM-CSF, matrilysin,
neuroendocrine protein
7B2, placental protein 11, gelsolin, M-CSF, transcobalamin I, lactase-
phlorizin hydrolase, elastase
2B, pepsinogen A, MIP 1-13, prolactin, trypsinogen II, gastrin-releasing
peptide II, atrial natriuretic
factor, secreted alkaline phosphatase, pancreatic a-amylase, secretogranin I,
p-casein, serotransferrin,
tissue factor pathway inhibitor, follitropin [3-chain, coagulation factor XII,
growth hormone-releasing
factor, prostate seminal plasma protein, interleukins (e.g., 2, 3,4, 5, 9,
11), inhibin (e.g., alpha chain),
angiotensinogen, thyroglobulin, IC heavy or light chains, plasminogen
activator inhibitor-1, lysozyme
C, plasminogen activator, antileukoproteinase 1, statherin, fibulin-1, isofonn
B, uromodulin,
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thyroxine-binding globulin, axonin-1, endometrial a-2 globulin, interferon
(e.g., alpha, beta, gamma),
13-2-microglobulin, procholecystokinin, progastricsin, prostatic acid
phosphatase, bone sialoprotein II,
colipase, Alzheimer's amyloid A4 protein, PDGF (e.g., A or B chain),
coagulation factor V,
triacylglycerol lipase, haptoglobuin-2, corticosteroid-binding globulin,
triacylglycerol lipase,
prorelaxin H2, follistatin 1 and 2, platelet glycoprotein IX, GCSF, VEGF,
heparin cofactor II,
antithrombin-III, leukemia inhibitory factor, interstitial collagenase,
pleiotrophin, small inducible
cytokine Al, melanin-concentrating hormone, angiotensin-converting enzyme,
pancreatic trypsin
inhibitor, coagulation factor VIII, a-fetoprotein, a-lactalbumin, senogelin
II, kappa casein, glucagon,
thyrotropin beta chain, transcobalamin II, thrombospondin 1, parathyroid
hormone, vasopressin
copeptin, tissue factor, motilin, MPIF-1, kininogen, neuroendocrthe convertase
2, stem cell factor
procollagen al chain, plasma kallikrein keratinocyte growth factor, as well as
any other secreted
hormone, growth factor, cytokine, enzyme, coagulation factor, milk protein,
immunoglobulin chain,
and the like.
[00210] In some embodiments, other secretory signal peptides encoded by the
rAAV genome and in
the rAAV vector as disclosed herein can be selected from, but are not limited
to, the secretory signal
sequences from prepro-cathepsin L (e.g., (IenBank Accession Nos. KHRTL,
NP_037288;
NP 034114, AAB81616, AAA39984, P07154, CAA68691; the disclosures of which are
incorporated
by reference in their entireties herein) and prepro-alpha 2 type collagen
(e.g., (ienBanIc Accession
Nos. CAA98969, CAA26320, CGHU2S, NP_000080, BAA25383, P08123; the disclosures
of which
are incorporated by reference in their entireties herein) as well as allelic
variations, modifications and
functional fragments thereof (as discussed above with respect to the
fibronectin secretory signal
sequence). Exemplary secretory signal sequences include for preprocathepsin L
(Rattus norvegicus,
IVITPLLLLAVLCLGTALA [SEQ ID NO: 27]; Accession No. CAA68691) and for prepro-
alpha 2
type collagen (Homo sapiens, MLSFVDTRTLLLLAVTLCLATC [SEQ ID NO: 28]; Accession
No.
CAA98969). Also encompassed are longer amino acid sequences comprising the
full-length secretory
signal sequence from preprocathepsin L and prepro-alpha 2 type collagen or
functional fragments
thereof (as discussed above with respect to the fibronectin secretory signal
sequence).
[00211] In some embodiments, the secretory signal peptide is derived in part
or in whole from a
secreted polypeptide that is produced by liver cells. In some embodiments, a
secretory signal peptide
can further be in whole or in part synthetic or artificial. Synthetic or
artificial secretory signal peptides
are known in the art, see e.g., Barash et al., "Human secretory signal peptide
description by hidden
Markov model and generation of a strong artificial signal peptide for secreted
protein expression,"
Biochem. Biophys. Res,, Comm. 294:835-42 (2002); the disclosure of which is
incorporated herein in
its entirety. In particular embodiments, the secretory signal peptide
comprises, consists essentially of,
or consists of the artificial secretory signal: MWWRLWWLLLLLLLLWPMVVVA (SEQ ID
NO: 29)
or variations thereof having 1, 2, 3, 4, or 5 amino acid substitutions
(optionally, conservative amino
acid substitutions, conservative amino acid substitutions are known in the
art).
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1002121 Exemplary signal peptides for use in the methods and compositions as
disclosed herein can
be selected from any signal peptide disclosed in Table 2, or fimctional
variants thereof. Exemplary
signal peptides are Fibronectin (FN1), or AAT. In some embodiments of the
methods and
compositions disclosed herein, the rAAV vector composition comprises the
nucleic acid encoding a
secretory signal peptide, e.g., encoding a secretory signal peptide selected
from an AAT signal
peptide (e.g., SEQ ID NO: 17), a fibronectin signal peptide (FN1) (e.g., SEQ
ID NO: 18-21), a GAA
signal peptide, an hIGF2 signal peptide (e.g., SEQ ID NO: 22) or an active
fragment thereof having
secretory signal activity, e.g., a nucleic acid encoding an amino acid
sequence that has at least about
75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ
ID NOs: 17-21
1002131 In some embodiments of the methods and compositions as disclosed
herein, the nucleic acid
encoding the secretory signal is selected from any of SEQ ID NO: 17, 81-21, 22-
26, or a nucleic acid
sequence at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence identity to
any of SEQ ID NOs: 17 or 22-26.
1002141 In some embodiments, one can readily substitute a FN1 or AAT signal
peptide with any
signal peptide, including signal peptides for over liver expressed proteins,
or signal peptides disclosed
in 62,937,556, filed on November 19, 2019, or PCT/US19/61653 filed on November
15, 2019.
1102151 Fibronectin secretory signal peptide:
1002161ln some embodiments, the secretory signal peptide is a fibronectin
secretory signal peptide,
which term includes modifications of naturally occurring sequences (as
described in more detail
below).
1002171 In some embodiments, the secretory signal peptide is a fibronectin
signal peptide, e.g., a
signal sequence of human fibronectin or a signal sequence from rat
fibronectin. Fibronectin (FN1)
signal sequences and modified FN1 signal peptides encompassed for use in the
rAAV genome and
rAAV vectors described herein are disclosed in US patent 7,071,172, which is
incorporated herein in
its entirety by reference, and in Table 3 of provisional application
62/937,556, filed on November 19,
2019. Examples of exemplary fibronectin secretory signal sequences include,
but are not limited to
those listed in Table 1 of US patent 7,071,172, which is incorporated herein
in its entirety by
reference.
1002181 Table 2: Exemplary Fibronectin (FN1) secretory signal peptides
Species Secretory Signal sequence
Nucleic acid sequence
H. Sapiens IVILRGPGPGLLLLAVQCLGTAV ATG CTT AGO (JOT CCG GGG CCC GGG CTG
PSTGA (SEQ ID NO: 20)
CTG CTG CTG GCC GTC CAG TGC CTG GGG
ACA GCG GTG CCC TCC ACG GGA GCC
(SEQ ID NO: 25)
R. MLRGPGPGRLLLLAVLCLGTSV 5'-
ATGCTCAGGGGTCCGGGACCCGGGCGGCT
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Norvegieus RCTETGKSKR (SEQ ID NO: 18) GCTGCTGCTAGCAGTCCTGTGCCTGGGGAC
ATCGGTGCGCTGCACCGAAACCGGGAAGA
GCAAGAGG-3 (SEQ ID NO: 23) (nucleotides
208-303)
R. MLRGPGPGRLLLLAVLCLGTSV 5'-ATG CTC AGO GGT
CCG GGA CCC GGG
Nolvegicus RCTETGKSKR t LALQIV
COO CTG CTG CTG CTA GCA GTC CTG TGC
(SEQ ID NO: 19)
CTG GGG ACA TCG GTG CGC TGC
ACC GAA ACC GGG AAG AGC AAG AGO
CAL) OCT CAL) CAA ATC GTG-3'. (SEQ ID
NO: 24) (t denotes the cleavage site)
X laevis ATG CGC CGG (JUG GCC CTG ACC GGG CTG
MRRGALTGLLLVLCLSVVLRA CTC CTG GTC CTG TGC CTG AGT GTT GTG
APSATSKICRR (SEQ ID NO: 21) CTA CUT GCA GCC CCC TCT GCA ACA AGC
AAG AAG CGC AGO (SEQ ID NO: 26)
1002191An exemplary nucleotide sequence encoding the fibronectin secretory
signal sequence of
Ramis norvegicus is found at GenBank accession number X15906 (the disclosure
of which is
incorporated herein by reference). As yet another illustrative sequence, the
nucleotide sequence
encoding the secretory signal peptide of human fibronectin 1, transcript
variant 1 (Accession No.
NM_002026, nucleotides 268-345; the disclosure of Accession No. NM_002026 is
incorporated
herein by reference in its entirety). Another exemplary secretory signal
sequence is encoded by the
nucleotide sequence encoding the secretory signal peptide of the Xenopus
laevis fibronectin protein
(Accession No. M77820, nucleotides 98-190; the disclosure of Accession No.
M77820 incorporated
herein by reference in its entirety).
1002201ln another embodiment, the fibronectin signal sequence (FN1,
nucleotides 208-303,5'-ATG
CTC AGO GUT CCG (iGA CCC GGG CGG CTG CTG CTG CTA GCA GTC CTG TGC CTG
GGG ACA TCG GTG CGC TGC ACC GAA ACC GGG AAG AGC AAG AGO-3', SEQ ID NO: 23)
was derived from the rat fibronectin mRNA sequence (Genbank accession #X15906)
and codes for
the following peptide signal sequence: Met Leu Arg Gly Pro Gly Pro Gly Arg Leu
Leu Leu Leu Ala
Val Leu Cys Leu Gly Thr Ser Val Arg Cys Thr Glu Thr Gly Lys Ser Lys Arg (SEQ
ID NO: 18). In
some embodiments of the methods and compositions disclosed herein, a
recombinant AAV vector
comprises a heterologous nucleic acid sequence that encodes a secretory signal
peptide which is a
fibronectin signal peptide (FN1) or an active fragment thereof having
secretory signal activity (e.g., a
FN1 signal peptide has the sequence of any of SEQ ID NO: 18-21, or an amino
acid sequence at
having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence identity to any
of SEQ ID NOs: 18-21), and the heterologous nucleic acid sequence encodes a
IGF2 targeting peptide
selected from any of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8
or SEQ ID NO:
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9, or a IGF2 peptide having at least about 75%, or 809o, or 85%, or 90%, or
95%, or 98%, or 99%
sequence identity to SEQ ID NOs: 5-9. In some embodiments of the methods and
compositions
disclosed herein, a recombinant AAV vector comprises a heterologous nucleic
acid sequence that
encodes a secretory signal peptide is AAT signal peptide or an active fragment
thereof having
secretory signal activity, (e.g., a AAT signal peptide has the sequence of SEQ
ID NO: 17, or an amino
acid sequence at having at least about 75%, or 80%, or 85%, or 90%, or 95%, or
98%, or 99%
sequence identity to SEQ ID NO: 17), and the heterologous nucleic acid
sequence encodes a IGF2
targeting peptide selected from any of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8
or SEQ ID NO: 9, or a IGF2 peptide having at least about 75%, or 80%, or 85%,
or 90%, or 95%, or
98%, or 99% sequence identity to SEQ ID NOs: 5-9.
1002211 Those skilled in the art will appreciate that the secretory signal
sequence may encode one,
two, three, four, five or all six or more of the amino acids at the C-terminal
side of the peptidase
cleavage site (identified by an t) (see e.g., SEQ ID NO: 19 and 24 in Table
2). Those skilled in the
art will appreciate that additional amino acids (e.g., 1, 2, 3,4, 5, 6 or more
amino acids) on the
carboxy-terminal side of the cleavage site may be included in the secretory
signal sequence.
1002221 In some embodiments of the methods and compositions disclosed herein,
a recombinant AAV
vector comprises, or consist of, located between the 5' ITR and the 3' ITR, a
heterologous nucleic
acid sequence that encodes a secretory signal peptide and nucleic acid
encoding a hGAA polypeptide,
where the nucleic acid sequence that encodes the signal sequence is selected
from any of: an AAT
signal peptide (e.g., SEQ ID NO: 17), a fibronectin signal peptide (FN1)
(e.g., SEQ ID NO: 18-21), a
cognate GAA signal peptide (SEQ ID NO: 175), an hIGF2 signal peptide (e.g.,
SEQ ID NO: 22), a
IgG1 leader peptide (SEQ ID NO: 177), wtIL2 leader peptide (SEQ ID NO: 179),
mutant IL2 leader
peptide (SEQ ID NO: 181) or an active fragment thereof having secretory signal
activity, e.g., a
nucleic acid encoding an amino acid sequence that has at least about 75%, or
80%, or 85%, or 90%, or
95%, or 98%, or 99% sequence identity to SEQ ID NOs: 17-22, 175, 177, 179 or
181, and where the
nucleic acid encoding the signal peptide is located 5' of a nucleic acid
encoding a hGAA polypeptide
as disclosed herein, and where the nucleic acid encoding the signal sequence
and the hGAA
polypeptide are operatively linked to any LSP disclosed herein in Table 4, or
a functional variant
thereof.
1002231). In embodiments of the invention, the functional fragment has at
least about 50%, 70%,
80%, 90% or more secretory signal activity as compared with the sequences
specifically disclosed
herein or even has a greater level of secretory signal activity.
1002241 Peptidase cleavage sites
1002251 In some embodiments, one or more exogenous peptidase cleavage site may
be inserted into
the secretory signal peptide - GAA fusion polypeptide, e.g., between the
secretory signal peptide and
the GAA polypeptide. In particular embodiments, an autoprotease (e.g., the
foot and mouth disease
virus 2A autoprotease) is inserted between the secretory signal peptide and
the GAA polypeptide or
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IGF2-GAA fusion polypeptide. In other embodiments, a protease recognition site
that can be
controlled by addition of exogenous protease is employed (e.g., Lys¨Arg
recognition site for trypsin,
the Lys¨Arg recognition site of the Aspergillus KEX2-like protease, the
recognition site for a
metalloprotease, the recognition site for a serine protease, and the [lice).
Modification of the GAA
polypeptide to delete or inactivate native protease sites is encompassed
herein and disclosed in U.S.
Provisional Application 62,937,556, filed on November 19, 2019 and
International Application
PCT/U519/61653, filed Nov 15, 2019.
C. IGF2 Targeting Peptide Sequence
1002261 In one embodiment, the rAAV genome comprises a heterologous nucleic
acid that encodes
a targeting peptide (TIP) fused to the GAA polypeptide. In some embodiments,
the targeting peptide is
a ligand for an extracellular receptor, wherein the targeting peptide binds an
extracellular domain of a
receptor on the surface of a target cell and, upon internalization of the
receptor, permits localization of
the polypeptide in a human lysosome. In one embodiment, the targeting peptide
includes a urokinase-
type plasminogen receptor moiety capable of binding the cation-independent
mannose-6-phosphate
receptor. In some embodiments, the targeting peptide incorporates one or more
amino acid sequences
of a IGF2 targeting peptide.
1002271 In some embodiments, the IGF2 targeting peptide as disclosed herein
comprises at least part
of a ligand for an extracellular receptor, for example, the IGF2 targeting
peptide binds to human
cation-independent mannose-6-phosphate receptor (CI-MPR) or the IGF2 receptor.
1002281 IGF2 is also known by alias; chromosome 11 open reading frame 43,
insulin-like growth
factor 2, IGF-II, FLJ44734; IGF2, somatomedin A and preptin. The mRNA of wild-
type human IGF2
sequence is corresponds to:
GCTTACCGCCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTC
TGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGT
GGCATCGTTGAGGAGTGCTGYITCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGT
GCTACCCCCGCCAAGTCCGAG (SEQ ID NO: 1). The full length IGF2 protein (including
the
IGF2 targeting sequence) is encoded by the nucleic acid sequence of NM
000612.6, and encodes the
full length IGF2 protein NP_000603.1.
1002291 The mature human IGF2 targeting peptide is shown below:
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEEC
CFRSCDLALLETYCATPAKSE(SEQIDNO: 5)
1002301 The coding sequence of human IGF2 is also disclosed in US patent
8,492,388 (see e.g.,
FIG. 2) which is incoiporated herein in its entirety by reference. IGF2
protein is synthesized as a pre-
pro-protein with a 24 amino acid signal peptide at the amino terminus and a 89
amino acid carboxy
terminal region both of which are removed post-translationally, reviewed in
O'Dell et al. (1998) hit. J.
Biochem Cell Biol. 30(7):767-71. The mature protein is 67 amino acids. A
Leishrnania codon
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optimized version of the mature IGF2 is disclosed in US patent 8,492,388 (see,
e.g, FIG. 3 of
8,492,388) (Langford et al. (1992) Exp. Parasitol. 74(3):360-1). Additional
cassettes containing a
deletion of amino acids 1-7 or 2-7 of the mature polypeptide (A1-7),
alteration of residue 27 from
tyrosine to leucine (Y27L) or both mutations (A1-7,Y27L or A2-7,Y27L) were
made to produce IGF-
2 cassettes with specificity for only the desired receptor as described below.
Accordingly, in some
embodiments, the IGF2 targeting sequence can be selected from any of:
wildtype, Y27L, A1-7, A2-7
and Y27L-A1-7, Y27L-A2-7, V43M, Y27L-V43M, Y27L-A1-7-V43M, Y27L-A2-7-V43M IGF2
variants are encompassed for use herein.
1002311 Exemplary IGF2 targeting peptide for use in the methods and
compositions herein are
disclosed in U.S. Provisional Application 62,937,556, filed on November 19,
2019 and International
Application PCT/US19/61653, filed Nov 15, 2019, and International application
PCT/US19/61701,
filed November 15, 2019, each of which are incorporated herein in their
entirety by reference.
1002321 In some embodiments, an IGF2 targeting peptide for use in the methods
and compositions
herein can have a modification of any one or more of: E6R, F265, Y27L, V43L,
F48T, R495, S501,
A54R, L55R, K65R, as disclosed in US application 2019/0343968, which is
incorporated herein in its
entirety. In some embodiments, the IGF2 targeting peptide has a modification
of V43M in addition to
one or more modifications selected from: E6R_, F26S, Y27L, V43L, F48T, R495,
S50I, A54R, L55R
and K65R. In some embodiments, the IGF2 targeting peptide has a A1-7 or A2-7
modification in
addition to one or more modifications selected from: E6R, F26S, Y27L, V43L,
F48T, R495, 550I,
A54R, L55R and K65R. In some embodiments, the IGF2 targeting peptide has a A1-
7 or A2-7
modification, a V43M modification, and one or more modifications selected
from: E6R, F265, Y27L,
V43L, F48T, R495, 5501, A54R, L55R and K65R.
1002331 In particular embodiments, the IGF2 targeting peptide comprises a
modification at valine
43, where valine is modified to a met (V43M), such that translation initiation
starts at amino acid 43.
A IGF2 targeting peptide with a modification of V43M encompassed for use
herein as a targeting
peptide or IGF2 targeting peptide binds the cation-independent mannose-6-
phosphate receptor. In
alterative embodiments, the IGF2 targeting peptide is delta 1-42 of IGF2 with
V43 changed to an Met
IGF2-A1-42 (SEQ ID NO: 8) or IGF2-V43M (SEQ ID NO:9).
1002341 In some embodiments, the rAAV genome comprises a nucleic acid encoding
an IGF2-GAA
fusion protein, where the nucleic acid encoding the mature IGF2 targeting
peptide (SEQ ID NO: 5) or
a IGF2 targeting peptide variant (e.g., SEQ ID NO: 6 (IGF2-A2-7); SEQ ID NO: 7
(IGF2-A1-7); SEQ
ID NO: 8 (IGF2--A1-42), SEQ ID NO: 9 (IGF2-V43M)) or sequences having at least
85%, or 90% or
95% sequence identity to SEQ ID NO: 5-9, is fused to the 5' end of nucleic
acid encoding the GALA
protein, fusion proteins (e.g., IGF2-GAA fusion polypeptides) are created that
can be taken up by a
variety of cell types and transported to the lysosome. Alternatively, a
nucleic acid encoding a
precursor IGF2 polypeptide can be fitsed to the 3' end of a GAA gene; the
precursor includes a
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carboxy-terminal portion that is cleaved in mammalian cells to yield the
mature IGF2 polypeptide, but
the IGF2 targeting peptide is preferably omitted (or moved to the 5' end of
the GAA gene). This
method has numerous advantages over methods involving glycosylation including
simplicity and cost
effectiveness, because once the protein is isolated, no further modifications
need be made.
[00235] In some embodiments, the IGF2 targeting peptide encompassed for use
herein is described
US patents 7,785,856 and 9,873,868 which are each incorporated herein in their
entirety by reference.
(I) Deletion mutants of IGF2:
[00236] In some embodiments, the IGF2 targeting peptide is a modified or
truncated IGF2 targeting
peptide (also referred to as a deletion mutant of IGF2), as disclosed in
International Application
PCT/US19/61701, filed Nov 15, 2019, which is incorporated herein in its
entirety by reference. For
example, in some embodiments, the IGF2 targeting peptide comprises a V43M
modification and also
any deletion of one or more amino acids from amino acid 1-42. For example, in
some embodiments of
the methods and compositions as disclosed herein, the IGF2 targeting peptide
comprises V43M and
further comprises one or more deletions selected from any of: A1-3, A14, A1-5,
A1-6, A1-8, A1-9,
A1-10, A1-11, A1-12, A1-13, A1-14, A1-15, A1-16, A1-17, A1-18, A1-19, A1-20,
A1-21, A1-22, A1-23,
A1-24, A1-25, A1-26, AI-27, A1-28, A1-29, A1-30, A1-31, A1-32, A1-33, A1-34,
A1-35, A1-36, A1-37,
A1-38, A1-39, A1-40, A1-41 or A1-42 of SEQ ID NO: 5 and wherein residue 43 of
SEQ ID NO: 5 is a
methionine (V43M). In some embodiments of the methods and compositions as
disclosed herein, the
IGF2 targeting peptide comprises V43M and further comprises a A1-7 deletion
(IGF2-A1-7,V43M).
[00237] In some embodiments of the methods and compositions as disclosed
herein, the lysosomal
IGF2 targeting peptide further comprises one or more modifications selected
from any of. A2-3, A2-4,
A2-5, A2-6, A2-8, A2-9, A2-10, A2-11, A2-12, A2-13, A2-14, A2-15, A2-16, A2-
17, A2-18, A2-19, A2-
20, A2-21, A2-22, A2-23, A2-24, A2-25, A2-26, A2-27, A2-28, A2-29, A2-30, A2-
31, A2-32, A2-33,
A2-34, A2-35, A2-36, A2-37, A2-38, A2-39, A2-40, A2-41 or A2-42 of SEQ ID NO:
5 and wherein
residue 43 of SEQ ID NO: 5 is a methionine (V43M). In some embodiments of the
methods and
compositions as disclosed herein, the IGF2 targeting peptide comprises V43M
and further comprises
a A2-7 deletion (IGF2-A2-7,V43M).
[00238] In some embodiments, a IGF2 targeting peptide for fusion to a GAA-
polypeptide can
comprise amino acids 8-28 and 41-61 of IGF2. In some embodiments, these
stretches of amino acids
can be joined directly or separated by a linker. Alternatively, amino acids 8-
28 and 41-61 can be
provided on separate polypeptide chains. In some embodiments, amino acids 8-28
of IGF2, or a
conservative substitution variant thereof, could be fused to GAA polypeptide
to express a IGF2-GAA
fusion protein from the rAVV vector, and a separate rAAV vector could express
IGF2 amino acids
41-61, or a conservative substitution variant thereof.
[00239] In order to facilitate proper presentation and folding of the IGF2
targeting peptide, longer
portions of IGF2 proteins can be used. For example, an IGF2 targeting peptide
including amino acid
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residues 1-67, 1-87, or the entire precursor form can be used.
1002401 In some embodiments, the IGF2 targeting peptide is a nucleic acid
sequence that encodes an
IGF2 targeting peptide of any of the following: residue 1 followed by residues
8-67 of wild-type
mature human insulin-like growth factor II (IGF2) of SEQ ID NO: 5 (i.e., SEQ
ID NO: 6; i.e., IGF2-
delta 2-7); residues 8-67 of wild-type mature human insulin-like growth factor
II (IGF2) of SEQ ID
NO: 5 (i.e., SEQ ID NO: 7; IGF2-delta 1-7) or residues 43-67 of wild-type
mature human insulin-like
growth factor II (IGF2) of SEQ ID NO: 5 (i.e., IGF2-V43M (SEQ ID NO: 9) or IGF-
delta 1-42 (SEQ
ID NO: 8).
1002411 hi some embodiments of the methods and compositions as disclosed
herein, the IGF2
targeting peptide is a nucleic acid sequence selected from any nucleic acid
sequence comprising any
of: SEQ ID NO: 2 (i.e., IGF2-delta 2-7); SEQ ID NO: 3 (i.e., IGF2-delta 1-7)
or SEQ ID NO: 4 (i.e.,
IGF2-V43M) or a sequence at least sequence at least 85%, 90%, 95%, 96%, 97%,
98% or 99%
sequence identity thereto.
1002421 In some embodiments of the methods and compositions as disclosed
herein, the
IGF2(V43M) sequence is a nucleic acid sequence encoding a IGF2(V43M) sequence
of any of SEQ
ID NO: 65 (IGF2A2-7V43M) or an amino acid sequence having at least 85%, or
90%, or 95% or
96%, or 97%, or 98% or 99% or 100% identity to SEQ ID NO: 65, or SEQ ID NO: 66
(IGFA1-
7V43M) or an amino acid sequence having at least 85%, or 90%, or 95% or 96%,
or 97%, or 98% or
99% or 100% identity to SEQ ID NO: 66.
Table 3: Exemplary nucleic acid sequences encoding IGF2 targeting peptide:
IGF2 targeting Sequence
peptide
IGF2-delta 2-7 GC11CTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGA
(IGFA2-7)
CCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCC
GTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGG
AGACGTACTGTGCTACCCCCGCCAAGTCCGAG) (SEQ ID NO: 2)
IGF2-delta 1-7 CTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCG
(IGFA1-7)
CGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTG
GCATCGTTGAGGAGTGCTGTTTICCGCAGCTGTGACCTGGCCCTCCTGGAG
ACGTACTGTGCTACCCCCGCCAAGTCCGAG (SEQ ID NO: 3)
IGF2-V43M
GCTTACCGCCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCT
CCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCOCAAGCC
GTGTGAGCCGTCGCAGCCGTGGCATCATGGAGGAGTGCTGITTCCGCAGC
TGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGA
G (SEQ ID NO: 4)
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10024311n some embodiments, in order to facilitate proper presentation and
folding of the IGF2
targeting peptide, longer portions of IGF2 proteins can be used. For example,
an IGF2 targeting
peptide including amino acid residues 1-67, 1-87, or the entire precursor form
can be used.
1002441 In some embodiments of the methods and compositions disclosed herein,
the recombinant
AAV comprises a heterologous nucleic acid sequence encoding a signal peptide-
GAA (SP-GAA)
fusion polypeptide further comprises a IGF2 targeting peptide located between
the secretory signal
peptide (SP) and the an alpha-glucosidase ((MA) polypeptide.
1002451 In some embodiments of the methods and compositions disclosed herein,
the recombinant
AAV vector comprises a heterologous nucleic acid sequence that encodes a IGF2
targeting peptide
which binds human cation-independent mannose-6-phosphate receptor (CI-MPR) or
the IGF2
receptor, for example, the heterologous nucleic acid sequence encodes a IGF2
targeting peptide
having the amino acid sequence of SEQ ID NO: 5 or comprises at least one amino
modification in
SEQ ID NO: 5 that binds to the IGF2 receptor. In some embodiments, the
recombinant AAV vector
comprises a heterologous nucleic acid sequence that encodes a IGF2 targeting
peptide that has at least
one amino modification in SEQ ID NO: 5 is a V43M amino acid modification (SEQ
ID NO: 8 or SEQ
ID NO: 9) or A2-7 (SEQ ID NO: 6) or A1-7 (SEQ ID NO: 7), or is a IGF2 peptide
having at least
about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to
SEQ ID NOs: 5-9.
1002461 In some embodiments of the methods and compositions disclosed herein,
the nucleic acid
encoding a IGF2 targeting peptide is selected from any of SEQ ID NO: 2 (IGF2-
A2-7), SEQ ID NO: 3
(IGF2-A1-7), or SEQ ID NO: 4 (IGF2 V43M), or a nucleic acid sequence at least
about 75%, or 80%,
or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to any of SEQ ID NOs:
2, 3 or 4.
1002471 In some embodiments of the compositions and methods described herein,
the IGF2
targeting peptide is a nucleic acid sequence that encodes any of residue 1
followed by residues 8-67
of wild-type mature human insulin-like growth factor II (IGF2) of SEQ ID NO: 5
(i.e., IGF2-delta 2-7
or IGF2A2-7; which corresponds to SEQ ID NO: 6); residues 8-67 of wild-type
mature human
insulin-like growth factor II (IGF2) of SEQ ID NO: 5 (i.e., IGF2-delta 1-7 or
IGF2A1-7, which
corresponds to SEQ ID NO: 7;) or residues 43-67 of wild-type mature human
insulin-like growth
factor II (IGF2) of SEQ ID NO: 5 (i.e., IGF2 delta 1-42 or IGF2A1-42, which
corresponds to SEQ ID
NO: 8). In some embodiments of the compositions and methods described herein,
the IGF2 targeting
peptide is a nucleic acid sequence that has a modification of amino acid
residue 43, for example
residue 43 is modified to a start codon, for example IGF2-V43M (corresponding
to SEQ ID NO: 9).
1002481 In some embodiments of the compositions and methods described herein,
the IGF2
targeting peptide is a nucleic acid sequence comprising any of: SEQ ID NO: 2
(i.e., IGF2-delta 2-7);
SEQ ID NO: 3 (i.e., IGF2-delta 1-7) or SEQ ID NO: 4 (i.e., IGF2-V43M).
1002491 In some embodiments of the compositions and methods described herein,
the fusion protein
comprising the GAA poly-peptide and a IGF2 targeting peptide comprises amino
acid residues 40-952
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or residues 70-952 of human acid alpha-glucosidase (GAA) polypeptide (SEQ ID
NO: 10) that is
attached to an IGF2 targeting peptide that comprises residue 1 followed by
residues 8-67 of wild-type
mature human insulin-like growth factor II (IGF2) (SEQ ID NO: 5), (that is -
residues 2-7 of mature
human IGF2 (SEQ ID NO:5) are not present), wherein the IGF2 targeting peptide
is linked to amino
acid residue 70 of human GAA (SEQ ID NO: 10).
1002501 In some embodiments of the compositions and methods described herein,
the fusion protein
comprising the GAA polypeptide and a IGF2 targeting peptide comprises amino
acid residues 40-952
or residues 70-952 of human acid alpha-glucosidase (GAA) polypeptide (SEQ ID
NO: 10) that is
attached to an IGF2 targeting peptide that comprises residues 8-67 of wild-
type mature human
insulin-like growth factor II (IGF2) (SEQ ID NO: 5), (that is - residues 1-7
of mature human IGF2
(i.e., YRPSE T; SEQ ID NO: 63) are not present), wherein the IGF2 targeting
peptide is linked to
amino acid residue 70 of human GAA (SEQ ID NO: 10).
1002511 In some embodiments of the compositions and methods described herein,
the fusion protein
comprising the GAA polypeptide and a IGF2 targeting peptide comprises amino
acid residues 40-952
or residues 70-952 of human acid alpha-glucosidase (GAA) (SEQ ID NO: 10) that
is attached to a
modified IGF2 targeting peptide that comprises residues 43-67 of wild-type
mature human insulin-
like growth factor II (IGF2) (SEQ ID NO: 5), (where residues 1-42 of mature
human IGF2 (SEQ ID
NO: 5) are not present), and where the IGF2 targeting peptide is linked to
amino acid residue 70 of
human GAA (SEQ ID NO: 10).
1002521In some embodiments of the methods and compositions disclosed herein, a
recombinant AAV
vector comprises a heterologous nucleic acid sequence that encodes an IGF2
peptide, where the IGF2
peptide sequence is SEQ ID NO: 8 or SEQ ID NO: 9, or a IGF2 peptide having at
least about 75%, or
80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 8
or 9.
(ii) Modified IGF2 targeting peptides and IGF2 homologues
1002531 In some embodiments, the nucleic acid encoding IGF2 can be modified to
diminish their
affinity for IGFBPs, and/or decreasing affinity for binding to IGF-I receptor,
thereby increasing
targeting to the lysosomes and increasing the bioavailability of the fused GAA-
polypeptide.
1002541 IGF2 targeting peptide preferably specifically targets and binds to
the M6P receptor.
Particularly useful are IGF2 targeting peptides which have mutations in the
IGF2 polypeptide that
result in a protein that binds the CI-MPR/M6P receptor with high affinity
while no longer binding the
other two receptors with appreciable affmity.
1002551 IGF2(V43M) targeting peptide is preferably targeted specifically to
the M6P receptor.
Particularly useful are IGF2(V43M) targeting peptides which have mutations in
the IGF2 polypeptide
that result in a protein that binds the CI-MPR/M6P receptor with high affinity
while no longer binding
the other two receptors with appreciable affinity.
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1002561 The IGF2(V43M) targeting peptide can also be modified to minimize
binding to serum
IGF-binding proteins (IGFBPs) (Baxter (2000) Am. J. Physiol Endocrinol Metab.
278(6):967-76) and
to IGF-I receptor, in order to avoid sequestration of IGF2 constructs. A
number of studies have
localized residues in IGF-1 and IGF2 necessary for binding to IGF-binding
proteins. Constructs with
mutations at these residues can be screened for retention of high affinity
binding to the M6P/IGF2
receptor and for reduced affinity for IGF-binding proteins. For example,
replacing Phe 26 of IGF2
with Ser is reported to reduce affinity of IGF2 for IGFBP-1 and -6 with no
effect on binding to the
M6P/IGF2 receptor (Bach etal. (1993) J. Biol. Chem. 268(13):9246-54). Other
substitutions, such as
Ser for Phe 19 and Lys for (Mu 9, can also be advantageous. The analogous
mutations, separately or in
combination, in a region of IGF-I that is highly conserved with IGF2 result in
large decreases in IGF-
BP binding (Magee et al. (1999) Biochemistry 38(48): 15863-70).
1002571 The IGF2 targeting peptide can also be modified to minimize binding to
serum IGF-binding
proteins (IGFBPs) and to IGF-I receptor, in order to avoid sequestration of
IGF2 constructs.
1002581 In some embodiments, a IGF2 targeting peptide is modified to be furin
resistant, i.e.,
resistant to degradation by furin protease, which recognizes Arg-X-X-Arg
cleavage sites. Such IGF2
targeting peptides are disclosed in US application 22012/0213762 which is
incorporated herein in its
entirety by reference. In some embodiments, a fitrin resistant IGF2 targeting
peptide for use in a
rAAV genome as described herein contains a mutation within a region
corresponding to amino acids
30-40 (e.g., 31-40, 32-40, 3340, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-
39, 34-39, 35-39, 36-
39, 37-40, 34-40) of SEQ ID NO: 5 (wt IGF2 targeting peptide) can be
substituted with any other
amino acid or deleted. For example, substitutions at position 34 may affect
furin recognition of the
first cleavage site. Insertion of one or more additional amino acids within
each recognition site may
abolish one or both furin cleavage sites. Deletion of one or more of the
residues in the degenerate
positions may also abolish both furin cleavage sites.
1002591 In some embodiments, a furin-resistant IGF2 targeting peptide contains
amino acid
substitutions at positions corresponding to Arg37 (R37) or Arg40 (R40) of SEQ
ID NO:5. In some
embodiments, a furin-resistant IGF2 targeting peptide contains a Lys (K) or
Ala (A) substitution at
positions Arg37 or Arg40 of SEQ ID NO: 5. Other substitutions are possible,
including combinations
of Lys and/or Ala mutations at both positions 37 and 40, or substitutions of
amino acids other than
Lys (K) or Ala (A). In some embodiments, the IGF2 targeting peptide
encompassed for use in the
rAVV genome as disclosed herein is IGFA2-7-K37, or IGFA2-7-K40 or IGFA1-7-K37
or IGFA1-7-
1(40, indicating that the IGF2 targeting peptides has a deletion of aa 2-7 or
1-7 and a modification of a
Arg (R) residue at position 37 to a lysine (i.e., R37K modification) or R4OK
respectively. In some
embodiments, the IGF2 targeting peptide encompassed for use in the rAVV genome
as disclosed
herein is IGFA2-7-K37-K40, or IGFA1-7-R37K-R4OK indicating that the IGF2
targeting peptides has
a deletion of residues 2-7 or residues 1-7 and a modification of a R residue
at position 37 and position
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40 to lysinines (R37K and R4OK). In some embodiments, the IGF2 targeting
peptide encompassed
for use in the rAVV genome as disclosed herein is selected from any of: IGFA2-
7-R37A, or IGFA2-
7-R40A or IGFA1-7-R37A or IGFA1-7-R40A, IGFA2-7-R37A-R40A, or IGFA1-7-R37A-
R40A.
Exemplary constructs for the IGF2 targeting peptide encompassed for use in the
rAVV genome as
disclosed herein are disclosed in US application 2012/0213762, which is
incorporated herein in its
entirety by reference.
[00260] In some embodiments, the furin-resistant IGF2 targeting peptide
suitable for the invention
may contain additional mutations. For example, up to 30% or more of the
residues of SEQ ID NO: 5
may be changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%
or more
residues may be changed). Thus, a furin-resistant IGF2 mutein suitable for the
invention may have an
amino acid sequence at least 70%, including at least 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical
to SEQ ID NO: 5.
[00261] Moreover, use of a IGF2 targeting peptide as disclosed herein is also
referred to in the art as
Glycosylation Independent Lysosomal Targeting (GILT) because the IGF2
targeting peptide replaces
M6P as the moiety targeting the lysosomes. Details of the GILT technology are
described in U.S.
Application Publication Nos. 2003/0082176, 2004/0006008, 2004/0005309,
2003/0072761,
2005/0281805, 2005/0244400, and international publications WO 03/032913, WO
03/032727, WO
02/087510, WO 03/102583, WO 2005/078077, the disclosures of all of which are
hereby incorporated
by reference.
[00262] Other modifications to the amino acid sequence of the IGF2 targeting
peptide for use in the
methods and compositions as disclosed herein are disclosed in US provisional
application
62,937,556, filed on November 19, 2019 and in PCT application PCT/US19/61653,
filed Nov 15,
2019, both of which are incorporated herein in their entirety by reference.
[00263] IGF2 binds to the IGF2/M6P and 1(1W-I receptors with relatively high
affinity and binds
with lower affinity to the insulin receptor. Substitution of IGF2 residues 48-
50 (Phe Arg Ser) with the
corresponding residues from insulin, (11w Ser Ile), or substitution of
residues 54-55 (Ala Leu) with the
corresponding residues from IGF-I (Arg Arg) result in diminished binding to
the IGF2/M6P receptor
but retention of binding to the IGF4 and insulin receptors (Sakano et al.
(1991) J. Biol. Chem.
266(31):20626-35).
[00264] IGF2 binds to repeat 11 of the cation-independent M6P receptor.
Indeed, a minireceptor in
which only repeat 11 is fused to the transmembrane and cytoplasmic domains of
the cation-
independent M6P receptor is capable of binding IGF2 (with an affinity
approximately one tenth the
affinity of the full length receptor) and mediating internalization of IGF2
and its delivery to lysosomes
(Grimme et al. (2000) J. Biol. Chem. 275(43):33697-33703). The structure of
domain 11 of the M6P
receptor is known (Protein Data Base entries IGPO and 1GP3; Brown et al.
(2002) EMBO J.
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21(5):1054-1062). The putative IGF2 binding site is a hydrophobic pocket
believed to interact with
hydrophobic amino acids of IGF2; candidate amino acids of IGF2 include leucine
8, phenylalanine
48, alanine 54, and leucine 55. Although repeat 11 is sufficient for IGF2
binding, constructs including
larger portions of the cation-independent M6P receptor (e.g. repeats 10-13, or
1-15) generally bind
IGF2 with greater affinity and with increased pH dependence (see, for example,
Linnell et at. (2001)
J. Biol. Chem. 276(26):23986-23991).
1002651 Substitution of IGF2 residues Tyr 27 with Leu, or Ser 26 with Phe
diminishes the affinity of
IGF2 for the IGF-I receptor by 94-, 56-, and 4-fold respectively (Torres et
al. (1995) J. Mol. Biol.
248(2):385-401). Deletion of residues 1-7 of human IGF2 resulted in a 30-fold
decrease in affinity for
the human IGF-I receptor and a concomitant 12-fold increase in affinity for
the rat IGF2 receptor
(Hashimoto et at. (1995) J. Biol. Chem. 270(30):18013-8). Truncation of the C-
terminus of IGF2
(residues 62-67) also appear to lower the affinity of IGF2 for the IGF-I
receptor by 5 fold (Roth et at.
(1991) Biochem. Biophys. Res. Conunun. 181(2):907-14).
1002661 Substitution of IGF2 residue phenylalanine 26 with serine reduces
binding to IGFBPs 1-5
by 5-75 fold (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Replacement
of IGF2 residues 48-
50 with threonine-serine-isoleucine reduces binding by more than 100 fold to
most of the IGFBPs
(Bach et al. (1993) J. Biol. Chem. 268(13):9246-54); these residues are,
however, also important for
binding to the cation-independent mannose-6-phosphate receptor. The Y27L
substitution that disrupts
binding to the IGF-I receptor interferes with formation of the ternary complex
with IGFBP3 and acid
labile subunit (Hashimoto et al. (1997) J. Biol. Chem. 272(44):27936-42); this
ternary complex
accounts for most of the IGF2 in the circulation (Yu et at. (1999) J. Clin.
Lab Anal. 13(4):166-72).
Deletion of the first six residues of IGF2 also interferes with IGFBP binding
(Luthi etal. (1992) Eur.
J. Biochem, 205(2):483-90),
1002671 Studies on IGF-I interaction with IGFBPs revealed additionally that
substitution of serine
for phenylalanine 16 did not affect secondary structure but decreased IGFBP
binding by between 40
and 300 fold (Magee et al. (1999) Biochemistry 38(48):15863-70). Changing
glutamate 9 to lysine
also resulted in a significant decrease in IGFBP binding. Furthermore, the
double mutant lysine
9/serine 16 exhibited the lowest affinity for IGFBPs, The conservation of
sequence between this
region of IGF-I and IGF2 suggests that a similar effect will be observed when
the analogous
mutations are made in IGF2 (glutamate 12 lysine/phenylalanine 19 serine).
1002681 In some embodiments, the IGF2(V43M) sequence comprises at least amino
acids 48-55; at
least amino acids 8-28 and 41-61; or at least amino acids 8-87, or a sequence
variant thereof (e.g.
R68A) or truncated form thereof (e.g. C-terminally truncated from position 62)
that binds the cation-
independent matmose-6-phosphate receptor.
1002691 In another embodiment of the invention, the rAAV genome encoding the
targeting peptide
(e.g., IGF2 targeting peptide) is inserted into the native (MA coding sequence
at the junction of the
mature 70/76 kDal polypeptide and the C-terminal domain, for example at
position 791. This creates a
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single chimeric polypeptide. In some embodiments, a protease cleavage site may
be inserted just
downstream of the targeting peptide (e.g., IGF2 targeting peptide).
[00270] In one embodiment, a targeting peptide, e.g., IGF2 targeting peptide
as defined herein, is
fused directly to the N- or C-terminus of the GAA polypeptide. In another
embodiment, a IGF2
targeting peptide is fused to the N- or C-terminus of the GAA polypeptide by a
spacer. In one specific
embodiment, a IGF2 targeting peptide is fused to the GAA polypeptide by a
spacer of 10-25 amino
acids. In another embodiment, a IGF2 targeting peptide is fused to the GAA
polypeptide by a spacer
including glycine residues.
[00271] In some embodiments, a IGF2 targeting peptide is fused to the GAA
polypeptide by a spacer
of at least 1, 2, or 3 amino acids. In some embodiments, the spacer comprises
amino acids GAP or
Gly-Ala-Pro (SEQ ID NO: 31), or an amino acid sequence at least 50% identical
thereto. In some
embodiments, the spacer is GGG or GA or AP, or GP or variants thereof. In some
embodiments, the
spacer is encoded by nucleic acids GGC GCG CCG (SEQ ID NO: 30).
[00272] In some embodiments, a IGF2 targeting peptide is fused to the GAA
polypeptide by a
spacer including a helical structure. In another specific embodiment, a IGF2
targeting peptide is fused
to the GAA polypeptide by a spacer at least 50% identical to the sequence
GGGTVGDDDDK (SEQ
ID NO: 35). In some embodiments of the methods and compositions as disclosed
herein, the spacer is
SEQ ID NO: 31 (encoded by nucleic acids of SEQ ID NO: 30). In some embodiments
of the methods
and compositions as disclosed herein, the spacer is selected from any of SEQ
ID NO: 31, SEQ ID
NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, or a sequence at least
sequence at least
85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
(iii) Alternative targeting peptides that hind to the Cation-independent 1116P
Receptor (CI-INPR).
[00273] In some embodiments, the targeting peptide is a lysosomal targeting
peptide or protein, or
other moiety other than the IGF2 targeting peptide disclosed herein that binds
to the cation
independent M6P/IGF2 receptor (CI-MPR) in a mannose-6-phosphate-independent
manner. The CI-
MPR also contains binding sites for at least three distinct ligands that can
be used as targeting
peptides. As disclosed herein, IGF2 ligand binds to CI-MPR with a dissociation
constant of about 14
nM at or about pH 7.4, primarily through interactions with repeat 11. The CI-
MPR is capable of
binding high molecular weight 0-glycosylated IGF2 forms. Accordingly, in some
embodiments, the
IGF2 targeting peptide can be post-transcriptionally modified to comprises 0-
glycosylation.
[00274] In an alternative embodiment, The targeting peptide that binds to CI-
MPR is retinoic acid.
Retinoic acid binds to the receptor with a dissociation constant of 2.5 nM.
Affinity photolabeling of
the cation-independent M6P receptor with retinoic acid does not interfere with
IGF2 or M6P binding
to the receptor, indicating that retinoic acid binds to a distinct site on the
receptor. Binding of retinoic
acid to the receptor alters the intracellular distribution of the receptor
with a greater accumulation of
the receptor in cytoplasmic vesicles and also enhances uptake of M6P modified
13-glucuronidase.
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Retiumic acid has a photoactivatable moiety that can be used to link it to a
therapeutic agent without
interfering with its ability to bind to the cation-independent M6P receptor.
1002751 The urokinase-type plasminogen receptor (uPAR) also binds CI-MPR with
a dissociation
constant of 9 pM. uPAR is a GPI-anchored receptor on the surface of most cell
types where it
functions as an adhesion molecule and in the proteolytic activation of
plasminogen and TGF-I3.
Binding of uPAR to the CI-M6P receptor targets it to the lysosome, thereby
modulating its activity.
Thus, fusing the extracellular domain of uPAR, or a portion thereof competent
to bind the cation-
independent M6P receptor, to a therapeutic agent permits targeting of the
agent to a lysosome.
D. Spacer and fusion junction of the GAA polypeptide
1002761Where GAA is expressed as a fusion protein with a secretory signal
peptide (e.g., SS-GAA
fusion polypeptide) or with a targeting peptide (i.e., SS-IGF2-GAA polypeptide
double fusion
polypeptide), the signal peptide or IGF2 targeting peptide can be fused
directly to the GAA
polypeptide or can be separated from the GAA polypeptide by a linker. An amino
acid linker (also
referred to herein as a "spacer") incorporates one or more amino acids other
than that appearing at that
position in the natural protein. Spacers can be generally designed to be
flexible or to interpose a
structure, such as an a-helix, between the two protein moieties.
1002771 Accordingly, in some embodiments of the methods and compositions
disclosed herein, a
recombinant AAV vector comprises a heterologous nucleic acid sequence encoding
an IGF2-GAA
fusion polypeptide, wherein the IGF2-GAA fusion protein further comprises a
spacer comprising a
nucleotide sequence of at least I amino acid in length, which is located N-
terminal to the GAA
polypeptide, and C-terminal to the IGF2 targeting peptide. In some embodiments
of the methods and
compositions disclosed herein, a recombinant AAV vector comprises a
heterologous nucleic acid
sequence that comprises a nucleic acid encoding a spacer of at least 1 amino
acids located between the
nucleic acid encoding the IGF2 targeting peptide and the nucleic acid encoding
the GAA polypeptide.
1002781 In one embodiment, the IGF2 targeting peptide is fused directly to the
N- or C-terminus of
the GAA polypeptide. In another embodiment, a IGF2 targeting peptide is fused
to the N- or C-
terminus of the GAA polypeptide by a spacer. In one specific embodiment, a
IGF2 targeting peptide
is fused to the GAA polypeptide by a spacer of 10-25 amino acids. In another
specific embodiment, a
IGF2 targeting peptide is fused to the GAA polypeptide by a spacer including
glycine residues. In
another specific embodiment, a IGF2 targeting peptide is fused to the GAA
polypeptide by a spacer
including a helical structure. In another specific embodiment, a IGF2
targeting peptide is fused to the
GAA polypeptide by a spacer at least 50% identical to the sequence GGGTVGDDDDK
(SEQ ID NO:
35).
1002791 In some embodiments, a spacer or linker can be relatively short, e.g.,
at least 1, 2, 3, 4 or 5
amino acids, or such as the sequence Gly-Ala-Pro (SEQ ID NO: 31) or Gly-Gly-
Gly-Gly-Gly-Pro
(SEQ ID NO: 32), or can be longer, such as, for example, 5-10 amino acids in
length or 10-25 amino
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acids in length. For example, flexible repeating linkers of 3-4 copies of the
sequence (GGGGS (SEQ
ID NO:33)) and a-helical repeating linkers of 2-5 copies of the sequence
(EAAAK (SEQ ID NO:34))
have been described (Arai et al. (2004) Proteins: Structure, Function and
Bioinformatics 57:829-838).
1002801The use of another linker, GGGTVGDDDDK (SEQ ID NO: 35), in the context
of an IGF2
fusion protein has also been reported (DiFalco et al. (1997) Biochern. J.
326:407413) and is
encompassed for use. Linkers incorporating an a-helical portion of a human
serum protein can be used
to minimize immunogenicity of the linker region.
1002811 In some embodiments, the spacer is encoded by nucleic acids GGC (KG
CCG (SEQ ID NO:
30) which encodes the amino acid spacer comprising amino acids GAP or Gly-Ala-
Pro (SEQ ID NO:
31).
1002821 The site of a fusion junction in the GAA polypeptide to fuse with
either the signal peptide (to
generate a SS-GAA fusion protein) or with the targeting peptide (e.g., to
generate a SP-IGF2-GAA
double fusion polypeptide) should be selected with care to promote proper
folding and activity of each
polypeptide in the fusion protein and to prevent premature separation of a
signal peptide from a GAA
polypeptide.
1002831 In some embodiments, a IGF2 targeting peptide is fused to the GAA
polypeptide by a spacer
including a helical structure. In another specific embodiment, a IGF2
targeting peptide is fused to the
GALA polypeptide by a spacer at least 50% identical to the sequence
GGGTVGDDDDK (SEQ ID NO:
35). In some embodiments of the methods and compositions as disclosed herein,
the spacer is SEQ ID
NO: 31 (encoded by nucleic acids of SEQ ID NO: 30). In some embodiments of the
methods and
compositions as disclosed herein, the spacer is selected from any of: SEQ ID
NO: 31, SEQ ID NO:
32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35.
1002841 Four exemplary strategies for creating a IGF2-GAA fusion protein can
be generated, which
are disclosed in provisional application 62,937,556, filed on November 19,
2019, and in
PCT/US19/61653, filed Nov 15, 2019, which are incorporated herein in their
entirety by reference.
1002851 In some embodiments, a targeting peptide (e.g., a IGF2 targeting
peptide) can be fused,
directly or by a spacer, to amino acid 40 or amino acid 70 of GAA, a position
permitting expression of
the protein, catalytic activity of the GALA protein, and proper targeting by
the IGF2 targeting peptide
as described herein in the Examples. Alternatively, a targeting peptide (e.g.,
a IGF2 targeting peptide)
can be fused at or near the cleavage site separating the C-terminal domain of
GAA from the mature
polypeptide. This permits synthesis of a GAA protein with an internal
targeting peptide (e.g., a IGF2
targeting peptide), which optionally can be cleaved to liberate the mature
polypeptide or the C-
terminal domain from the targeting domain, depending on placement of cleavage
sites. Alternatively,
the mature polypeptide can be synthesized as a fusion protein at about
position 791 without
incorporating C-terminal sequences in the open reading frame of the expression
construct.
[00286] In order to facilitate folding of the IGF2 targeting peptide, GAA
amino acid residues adjacent
to the fusion junction can be modified. For example, since it is possible that
GAA cysteine residues
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may interfere with proper folding of the targeting peptide (e.g., a IGF2
targeting peptide), the terminal
GAA cysteine 952 can be deleted or substituted with serine to accommodate a C-
terminal targeting
peptide (e.g., a IGF2 targeting peptide). The targeting peptide (e.g., a IGF2
targeting peptide) can also
be fused immediately preceding the final Cys952. The penultimate cys938 can be
changed to proline
in conjunction with a mutation of the final Cys952 to serine.
K CS sequence
10028711n some embodiments of the methods and compositions disclosed herein, a
recombinant AAV
vector comprises a heterologous nucleic acid sequence that further comprises
at collagen stability
(CS) sequence located 3' of the nucleic acid encoding the GAA polypeptide and
5' of the 3' ITR
sequence. In some embodiments, the rAAV genome disclosed herein comprises a
heterologous
nucleic acid sequence that can optionally comprise a Collagen stability
sequence (CS or CS 5), which
is positioned 3' of the GAA gene and 5' of a polyA signal. In some
embodiments, the CS sequence
can be replaced by a 3' UTR sequence as disclosed herein.
1002881 Exemplary collagen stability sequences include CCCAGCCCAC'TITTCCCCAA
(SEQ ID
NO: 65) or a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence
identity thereto.
An exemplary collagen stability sequence can have an amino acid sequence of
PSPLFP (SEQ ID
NO: 66) or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%
or 99% sequence
identity thereto. CS sequences are disclosed in Holick and Liebhaber, Proc.
Nat. Acad. Sci. 94: 2410-
2414, 1997 (See, e.g. Figure 3, p. 5205), which is incorporated herein its
entirety by reference.
F. Promoters
1002891 In some embodiments, to achieve appropriate levels of GAA expression,
the rAAV
genotype comprises a liver specific promoter (LSP). A LSP enables expression
of the operatively
linked gene in the liver, and can in some embodiments, be and inducible LSP.
In an embodiment, a
LSP is located upstream 5' and is operatively linked to the heterologous
nucleic acid sequence
encoding the GAA protein. Exemplary liver-specific promoters are disclosed
herein, and include for
example, the LSP comprising SEQ ID NO: 86, 91-96 or 146-150, or functional
variant or functional
fragment thereof, or any LSP listed in Table 4 herein, or a functional
fragment or functional variants
thereof. In some embodiments of the compositions and methods disclosed herein,
a liver-specific
promoter includes a liver-specific cis-regulatory element (CRE), a synthetic
liver-specific cis-
regulatory module (CRM) or a synthetic liver-specific promoter is selected
from any of SEQ ID NO:
270-341 (minimal LSP with CRM) or SEQ ID NO: 342-430 (synthetic liver specific
proximal
promoters) disclosed in Table 4 herein). In some embodiments, an rAAV vector
genome can include
one or more constitutive promoters, such as viral promoters or promoters from
mammalian genes that
are generally active in promoting transcription.
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(i) Synthetic Liver-specific promoters
1002901 In some embodiments of the methods and compositions as disclosed
herein, the promoter is
a liver specific promoter, and can be selected from promoters including, but
not limited to, those listed
in Table 4 disclosed herein or functional variants thereof, and or any
selected from Tables 4A and 4B
of U.S. provisional application 62,937,556, filed on November 19, 2019, or
functional variants thereof
1002911 While transthyretin promoter (FIR) (SEQ ID NO: 431) and 5P0412 (SEQ ID
NO: 91) and
5P0422 (SEQ ID NO: 92) are used as an exemplary liver specific promoters (see
Examples 1, 12 and
13) in the specification and Examples, one of ordinary skill in the art can
readily replace TER with
any liver specific promoter as disclosed herein in Table 4 or functional
variants thereof, and or any
selected from Tables 4A and 4B of U.S. provisional application 62,937,556,
filed on November 19,
2019, or functional variants thereof. A liver-specific promoter can comprise a
liver-specific cis-
regulatory element (CRE), a synthetic liver-specific cis-regulatory module
(CRM) or a synthetic liver-
specific promoter as disclosed herein, in Tables 4A and 413 of U.S.
provisional application
62,937,556, filed on November 19, 2019, or functional variants thereof.
1002921 Table 4 shows exemplary liver-specific promoters. The relatively small
size of liver-
specific promoters disclosed herein is advantageous because it takes up the
minimal amount of the
payload of the vector. This is particularly important when a LSP is used in a
vector with limited
capacity, such as an AAV-based vector,
1002931 Table 4: Exemplary LSP identified by SEQ ID NOs for use in the methods
and
compositions as disclosed herein
Table 4: Exemplary LSP
SEQ
SEQ ID
SEQ ID
NO:
Name of LSP ID Name of LSP NO Name of LSP
:
NO:
SP0269
270 CRM SP0107
330 CRM SP0396 384
(LVR 132 V1)
SP0270
271 CRM SP0109
331 CRM_5P0397 385
(LVR 132 V2)
SP0271
273 CRM SPO1 11
332 CRM_5P0398 386
(LVR_133 A 1 )
SP0272
274 CRM SP0112
333 CRM_SP0399 387
(LVR 133 V1)
388
SP0273
275 CRM SP0113
334 CRM_5P0403
(LVR_133_V2)
276 CRM SP0115
335 CRM SP0404 5P0256
389
277 CRM 390 SP0116
336 CRM_SP0405 5P0257
278 CRNI SP0121
337 CRNI SP0406 391 SP0258
338
279 CRM SP0124
CRM SP0407 392 SP0259
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CRM SP0127 339
280
(CRM- LVR 127)
CRM_SP0409 393 SP0264
CRM 5P0127A1
SP0265(LVR SP1
281
(CRM-
LVR_127 A 1) 340 CRNI_S P0411
394 31 Al)
CRM SP0127V1
282
SP0266(LVR SP1
(CRM LVR 127 V1) 86
CRNI_SP0412 395
31 VI)
CRM 5P0127V2
283
SP0267(LVR SP1
(CRIVI LVR 127 V2) 341 CRM_SP0413
396
31V2)
284 CRM SP0128 342 SP0107
397 5P0268
(LVR 132 Al)
CRM SP013 I
SP0269
285 343 SP0109
398
(CRM LVR 131)
(LVR 132 V 1)
CRM SP0132
5P0270
286 344 SP0111
399
(CRM I LVR 132)
(LVR 132 V2)
287 CRNI SP0133
5P0271
(CRM LVR 133) 345 SP0112
400
(LVR 133 Al)
288 CRNI SP0155 346 5P0113
401 SP0272
(LVR 133_V1)
289 CRM _5P0158 347 SP0115
402 SP0273
(LVR 133 V2)
290 CRIVI SP0163 348 5P0116
403 SP0368
291 CRM 5P0236 349 SP0121
404 SP0373
292 CR/VI SP0239 350 SP0124
5P0378
405
293 CRM SP0240 5P0127
406
SP0379
351 (LVR
SP127)
SP0127A1
294 CRNI SP0241 352 (LVR SP127 A 1
407 SP0380
)
SP0127V1
295 CRM _5P0242 353 (LVR SP127_V1
408 SP0381
)
296 SP0127V2
409
CRM SP0243 354 (LVR SPI27 V2
SP0272
)
(LVR_133 V1)
410
297 CRM _5P0244 355 SP0128
SP0273
(LVR_133_V2)
298 CRM SP0246 356 SP0131
411
SP0368
(LVR SP13 1)
299 CRM SP0247 357 SP0132
412
SP0373
(LVR SP132)
300 CRM SP0248 358 SP013-3
413
SP0378
(LVR SP133)
301 CHM SP0249 359 SP0155
414 SP0379
302 CR/VI SP0250 360 SP0158
415 SP0380
303 CRM _5P0251 361 SP0163
416 SP0381
304 CRM 5P0252 362 5P0236
417 SP0384
305 CRM _5P0253 93 5P0239
418 SP0388
306 CHM SP0254 95 5P0240
419 SP0396
307 CRNI SP0255 363 5P0241
420 SP0397
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308 CRM SP0256 364 SP0242
421 SP0398
309 CR/VI SP0257 365 SP0243
422 SP0399
310 CRM SP0258 366 SP0244
423 SP0403
311
CRM SP0259 96 SP0246
424 SP0404
312 CRM SP0264 367 SP0247
425 SP0405
CRM SP0265
313
426 SP0406
(CRM LVR 131 Al) 368 SP0248
CRM SP0266
314
(CRIVI LVR 131 V1) 369 SP0249
427 SP0407
315
CRM SP0267
(CRM_
LVR 13 l_V2) 370 SP0250
428 SP0409
CRM SP0268
316
(CRM¨
LVR 132 Al) 371 SP0251
429 SP0411
317
CRM SP0269
(CRM_
LVR 132_V1) 372 SP0252
91 SP0412
CRM SP0270
318
(CR/VI LVR 132 V2) 373 SP0253
92 SP0422
319
CRM SP0271
(CRM_
LVR 133_Al) 374 SP0254
146 SP0265-UTR
CRM SP0272
320
(CRM¨
LVR 133 VI) 375 SP0255
147 SP0239-UTR
321
CRM SP0273
(CRM_
LVR 133 V2) 376 SP0256
148 SP0240-UTR
322 CRM SP0368 377 SP0257
149 SP0246-UTR
323 CR/VI SP0373 378 SP0258
150 SP0131-A1-UTR
324 CRM SP0378 379 SP0259
430 SP0413
325 CRIVI SP0379 380 SP0264
431 =f 1K promoter
SP0265
326 CRM (LVR SP0380 94
, SP131_Al
432
LP1
)
327 CRM SP038 1 381 SP0266(LVR_SP
433 C -1E
131 Vi)
328 CRM SP0267(LVR_SP
SP0384 382 434 CBA
131 V2)
SP0268
329 CRM SP0388 383
435 TUG promoter
(LVR 132 Al)
(ii) Functional variants of Liver-Specific promoters
1002941 In some embodiments, the synthetic liver-specific promoter usefid in
the methods and
compositions as disclosed herein is a bi-specific, or tri-specific promoter as
defined herein. As an
illustrative example, a liver bi-specific promoter is active in the liver arid
one other tissue, for
example, the muscle. Additionally, another illustrative example of a liver bi-
specific promoter is
active in the liver and one other tissue, e.g., the brain. As an illustrative
example of a liver tri-specific
promoter is active in the liver and two other tissues, for example, the muscle
and brain. Additionally,
another illustrative example of a liver tri-specific promoter is active in the
liver and two other tissues,
such as, e.g., the kidney and muscle.
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[00295] In some embodiments, a synthetic liver specific promoter that is at
least 50%, 60%, 70%,
80%, 90% or 95% identical to any of SEQ ID NO: 86, 91-96, 146-150, 270-430
comprises a source
regulatory nucleic acid sequence which is preferentially active in liver, and
is also active to a lesser
extent (e.g., <50%, or about 49-40%, or about 39-30%, or about 29-20% or about
19-10% or <10% of
total expression) in a second type of cell or tissue, e.g., muscle or CNS.
[00296] In some embodiments, the promoter is a synthetic liver-specific
promoter comprising a
combination of the cis-regulatory elements (CREs) CRE0051 (SEQ ID NO: 97) and
CRE0042 (SEQ
ID NO: 104), or functional variants thereof. Functional variants thereof may
have a sequence that is
at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical thereto.
Typically, the CREs are operably linked to a promoter element. In some
preferred embodiments, the
liver-specific promoter comprises said CREs, or functional variants thereof,
in the order CRE0051
(SEQ ID NO: 97), CRE0042 (SEQ ID NO: 104), and then the promoter element
(order is given in an
upstream to downstream direction, as is conventional in the art).
[00297] The promoter element can be any suitable proximal promoter or minimal
promoter. In
some embodiments, the promoter element is a minimal promoter. Where the
promoter is a proximal
promoter, it is generally preferred that the proximal promoter is liver-
specific.
[00298] In some preferred embodiments, the promoter element is CRE0059 (SEQ ID
NO: 110), or a
functional variant thereof. CRE0059 is a proximal promoter, as is discussed
further below.
[00299] Thus, in one embodiment the promoter comprises the following
regulatory elements:
CRE0051 (SEQ ID NO: 97), CRE0042 (SEQ ID NO: 104) and CRE0059 (SEQ ID NO:
110), or
functional variants thereof.
[00300] Functional variants of CRE0051 (SEQ ID NO: 97) are regulatory elements
with sequences
which vary from CRE0051, but which substantially retain activity as liver-
specific CREs. It will be
appreciated by the skilled person that it is possible to vary the sequence of
a CRE while retaining its
ability to bind to the requisite transcription factors (TFs) and enhance
expression. A functional
variant can comprise substitutions, deletions and/or insertions compared to a
reference CRE, provided
they do not render the CRE substantially non-functional.
[00301] In some embodiments, a functional variant of CR E0051 can be viewed as
a CRE which,
when substituted in place of CRE0051 in a promoter, substantially retains its
activity. For example, a
liver-promoter which comprises a fiinctional variant of CRE0051 substituted in
place of CRE0051
preferably retains 80% of its activity, more preferably 90% of its activity,
more preferably 95% of its
activity, and yet more preferably 100% of its activity. For example,
considering promoter SP0412
(SEQ ID NO: 91) as an example, CRE0051 in SP0412 can be replaced with a
functional variant of
CRE0051, and the promoter substantially retains its activity. Retention of
activity can be assessed by
comparing expression of a suitable reporter under the control of the reference
promoter with an
otherwise identical promoter comprising the substituted CRE under equivalent
conditions.
[00302] In some embodiments the functional variant of CRE0051 comprises
transcription factor
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binding sites (TFBS) for the same liver-specific TFs as CRE0051. The liver-
specific TFBS present in
CRE0051, listed in the order in which they are present, are: HNF1 (SEQ ID NO:
98), HNF4 (SEQ ID
NO: 99), HNF3 (SEQ ID NO: 100), HNF1' (SEQ ID NO: 101) and HNF3' (SEQ ID NO:
102), see
Table 5. The functional variant of CRE0051 thus preferably comprises all of
these TFBS. Preferably,
they are present in the same order that they are present in CRE0051, i.e. in
the order HNF1 (SEQ ID
NO: 98), HNF4 (SEQ ID NO: 99), FINF3 (SEQ ID NO: 100), HNF1' (SEQ ID NO: 101)
and HNF3'
(SEQ ID NO: 102),. When the cis-regulatory element is associated with a
promoter and gene, this
order is preferably considered in an upstream to downstream direction (i.e. in
the direction from distal
from the transcription start site (TSS) to proximal to the TSS). Spacer
sequences may be provided
between adjacent TFBS. In some embodiments the TFBS may suitably overlap,
provided they remain
functional, i.e. overlapping sequences are both able to bind their respective
TFs to the extent required
to regulate expression.
1003031 In some embodiments the functional variant of CRE0051 (SEQ ID NO: 97)
comprises the
following TFBS sequences: GTTAA11111AAA (HNE1) (SEQ ID NO: 98), GTGGCCCTTGG
(HNF4) (SEQ ID NO: 99), TGTTTGC (HNF3) (SEQ ID NO: 100), TGGTTAATAATCTCA
(HNF1') (SEQ ID NO: 101) then ACAAACA (FINF3) (SEQ ID NO: 102), sequences
complementary
thereto, or functional variants of these TFBS sequences that maintain the
ability to bind to their
respective TF. These may be present in the same order as CRE0051, i.e. the
order in which they are
set out above. It is well-known in the art that there is sequence variability
associated with TFBS, and
that for a given TFBS there is typically a consensus sequence, from which some
degree of deviation is
typically present. Further information about the variation that occurs in a
TFBS can be illustrated
using a positional weight matrix (PWM), which represents the frequency with
which a given
nucleotide is typically found at a given location in the consensus sequence.
Details of TF consensus
sequences and associated PWMs can be found in, for example, the Jaspar or
Transfac databases
(http://jaspar.genereg.net/ and http://gene-
regulation.com/pub/databases.html). This information
allows the skilled person to modify the sequence in any given TFBS of a CRE in
a manner which
retains, and in some cases even increases, CRE functionality.
1111103041 In some embodiments, the functional variant of CRE0051 comprises
the sequence:
1003051 GTTAA _____________________ 111 11AAA-Na-GTGGCCCTTGG-Nb-TGTTTGC-Nc-
TGGTTAATAATCTCA-
Nd-ACAAACA (SEQ ID NO: 103), or a sequence that is at least 70%, 80%, 90%, 95%
or 99%
identical thereto, wherein Na, Nb, Nc, and Nd represent optional spacer
sequences. When present, Na
optionally has a length of from 10 to 26 nucleotides, preferably from 14 to 22
nucleotides, and more
preferably 18 nucleotides. When present, Nb optionally has a length of from 8
to 22 nucleotides,
preferably from 12 to 20 nucleotides, more preferably 16 nucleotides. When
present, Nc optionally
has a length of from 1 to 10 nucleotides, preferably 1 to 5 nucleotides, and
more preferably 2
nucleotides. When present, Nd suitably has a length of from 1 to 13
nucleotides, preferably from 2 to
9 nucleotides in length, and more preferably 5 nucleotides in length.
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[00306] In some embodiments, the CRE consists of SEQ ID No: 98-102 or a
functional variant
thereof.
[00307] It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleotide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 97-102 or a
functional variant
thereof fall within the scope of the invention. Single stranded nucleic acids
comprising the sequence
according to SEQ ID NO: 97 or 103 or a functional variant thereof also fall
within the scope of the
invention.
[00308] In some embodiments, the CRE comprising or consisting of CRE0051 (SEQ
ID NO: 97), or
a functional variant thereof, has a length of 200 or fewer nucleotides, 150 or
fewer nucleotides, 125 or
fewer nucleotides, or 100 or fewer nucleotides.
1003091 In some embodiments, the CRE comprising or consisting of CRE0042 (SEQ
ID NO: 104)
or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, or 100 or fewer nucleotides. Functional variants thereof
may have a sequence
that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical
thereto.
[00310] Functional variants of CRE0042 (SEQ ID NO: 104) are regulatory
elements with sequences
which vary from CRE0042, but which substantially retain their activity as
liver-specific CREs. It will
be appreciated by the skilled person that it is possible to vary the sequence
of a CRE while retaining
its ability to bind to the requisite transcription factors (TFs) and enhance
expression. A functional
variant can comprise substitutions, deletions and/or insertions compared to a
reference CRE, provided
they do not render the CRE substantially non-fiinctional.
[00311] In some embodiments, a functional variant of CRE0042 (SEQ ID NO: 104)
can be viewed
as a CRE which, when substituted in place of CRE0042 in a promoter,
substantially retains its
activity. For example, a promoter which comprises a functional variant of
CRE0042 substituted in
place of CRE0042 preferably retains 80% of its activity, more preferably 90%
of its activity, more
preferably 95% of its activity, and yet more preferably 100% of its activity
(compared to the reference
promoter comprising CRE0042 (SEQ ID NO: 104)). For example, considering
promoter SP0412 as
an example, CRE0042 (SEQ ID NO: 104) in 5P412 (SEQ ID NO: 91) can be replaced
with a
functional variant of CRE0042, and the promoter substantially retains its
activity. Retention of
activity can be assessed by comparing expression of a suitable reporter under
the control of the
reference promoter with an otherwise identical promoter comprising the
substituted CRE under
equivalent conditions.
[00312] In some embodiments it is preferred that the functional variant of
CRE0042 (SEQ ID NO:
104) comprises TFBS for the same liver-specific TFs as CRE0042. The liver-
specific TFBS present
in CRE0042, listed in the order in which they are present, are: HNF-3 (SEQ ID
NO: 106), C/EBP
(SEQ ID NO: 107), HNF-4 (SEQ ID NO: 108) and C/EBP' (SEQ ID NO: 109). The
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variant of CRE0042 thus preferably comprises all of these TFBS. Preferably,
they are present in the
same order that they are present in CRE0042, i.e. in the order HNF-3, C/EBP,
HNF-4 and then
C/EBP. When the cis-regulatory element is associated with a promoter and gene,
this order is
preferably considered in an upstream to downstream direction (i.e. in the
direction from distal from
the transcription start site (TSS) to proximal to the TSS). Spacer sequences
may be provided between
adjacent TFBS. In some embodiments the TFBS may suitably overlap, provided
they remain
functional, i.e. overlapping sequences are both able to bind their respective
TEs.
[00313] In some embodiments the functional variant of CRE042 (SEQ ID NO: 104)
comprises the
following TFBS sequences: GTTCAAACATG (IINF-3) (SEQ ID NO: 106), CTAATACTCTG
(C/EBP) (SEQ ID NO: 107), TGCAAGGGTCAT (IINF-4) (SEQ ID NO: 108), and
TTACTCAACA
(C/EBP) (SEQ ID NO: 109) and sequences complementary thereto, or functional
variants of these
TFBS sequences that maintain the ability to bind to their respective TF. These
may be present in the
same order as CRE0042, i.e. the order in which they are set out above. As
discussed above, it is well-
known in the art that there is sequence variability associated with TFBS, and
that for a given TFBS
there is typically a consensus sequence, from which some degree of deviation
is typically present.
[00314] In some embodiments of the invention, the functional variant of
CRE0042 comprises the
sequence:
[00315] GITCAAACATG-Na-CTAATACTCTG-Nb-TGCAAGGGTCAT-Nc-TTACTCAACA
(SEQ ID NO: 105) or a sequence that is at least 70%, 80%, 90%, 95% or 99%
identical thereto,
wherein Na, Nb and Nc represent optional spacer sequences. When present, Na
optionally has a
length of from 1 to 10 nucleotides, preferably from 1 to 5 nucleotides, and
more preferably 2
nucleotides. When present, Nb optionally has a length of from 1 to 10
nucleotides, preferably from 2
to 6 nucleotides, and more preferably 4 nucleotides. When present, Nc
optionally has a length of
from 8 to 23 nucleotides, preferably from 10 to 20 nucleotides, and more
preferably 15 nucleotides.
[00316] In some embodiments of the invention the cis-regulatory enhancer
element consists of
CRE0042 (SEQ ID NO: 104) or a functional variant thereof.
[00317] It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleotide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 104 or 105 or
a functional
variant thereof fall within the scope of the invention. Single stranded
nucleic acids comprising the
sequence according to SEQ ID NO: 104 or 105 or a functional variant thereof
also fall within the
scope of the invention.
In some embodiments, the CRE comprising or consisting of CRE0042 (SEQ ID NO:
104), or a
fmictional variant thereof, has a length of 200 or fewer nucleotides, 150 or
fewer nucleotides, 125 or
fewer nucleotides, 100 or fewer nucleotides, or 80 or fewer nucleotides.
[00318] In some embodiments, the CRE comprising or consisting of CRE0059 (SEQ
ID NO: 110)
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or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, or 100 or fewer nucleotides. Functional variants thereof
may have a sequence
that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical
thereto.
[00319] As discussed above, functional variants of CRE0059 (SEQ ID NO: 110)
substantially retain
the ability of CRE00059 to act as a liver-specific promoter element. For
example, when a functional
variant of CRE0059 is substituted into liver-specific promoter 5P0412, the
modified promoter retains
at least 80% of its activity, more preferably at least 90% of its activity,
more preferably at least 95%
of its activity, and yet more preferably 100% of the activity of 5P0412 (SEQ
ID NO; 91). Suitably
the functional variant of CRE0059 comprises a sequence which has at least 70
A, 80%, 90%, 95% or
99% identity to SEQ ID NO: 110.
[00320] CFtE0059 is a proximal promoter and comprises a TFBS for a liver-
specific TF, namely
HNF1, upstream of the TSS. The functional variant of CRE0059 thus preferably
comprises a TFBS
for HNF1 upstream of the TSS.
[00321] In some embodiments, a functional variant of CRE0059 comprises a
sequence which is at
least 70% identical to SEQ ID NO: 110 (preferably at least 80%, 90%, 95% or
99% identical to SEQ
ID NO: 110), which contains a TFBS for HNF1 (SEQ ID NO: 111), and which
contains a TSS
sequence (referred to as pl@SERPINA1 or pl@AFP) which is at least 80%, 90%,
95% or completely
identical to SEQ ID NO: 112 downstream of said TFBS for HNF1.
[00322] In some embodiments, a functional variant of CRE0059 comprises a
sequence which has at
least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO: 110, and which further
comprises a TFBS
comprising SEQ ID NO: 111 for 1-INF1 at or near position 24-36; and which
comprises the TSS
sequence which is at least 80%, 90%, 95% or completely identical to SEQ ID NO:
112 at or near
position 73-93, positions being numbered with reference to SEQ ID NO: 110. At
or near in the
present context suitably means within 10, 5,4, 3, 2, or 1 nucleotide of the
recited position with
reference to SEQ ID NO: 110. Suitable TFBS sequences are SEQ ID NOS 111 and
SEQ ID NO: 112,
but alternative TFBS sequences can be used.
[00323] In some embodiments, a promoter element comprising or consisting of
CRE0059 (SEQ ID
NO: 110) or a functional variant thereof has a length of 200 or fewer
nucleotides, 150 or fewer
nucleotides, 125 or fewer nucleotides, 110 or fewer nucleotides, or 95 or
fewer nucleotides.
1003241 In some embodiments the liver-specific promoter useful in the methods
and compositions
as disclosed herein comprises or consists of SEQ ID NO: 91, or a functional
variant thereof. In some
embodiments, functional variants may have a sequence that is at least 60%,
65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. The promoter having a
sequence
according to SEQ ID NO: 91 is referred to as 5P0412. The 5P0412 promoter is
particularly preferred
in some embodiments. This promoter has been found to be powerful and is also
very short, which is
advantageous in some circumstances.
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SP0265 (also known as SP131A1) and variants thereof
[00325] In some embodiments, the promoter is a synthetic liver-specific
promoter comprising a
combination of the CREs CRE0051 (SEQ ID NO: 97), CRE0058 (SEQ ID NO: 113),
CRE0065 (SEQ
ID NO: 117), and CRE0066 (SEQ ID NO: 122), or functional variants thereof.
Typically, the CREs
are operably linked to a promoter element. In some preferred embodiments, the
liver-specific
promoter comprises said CREs, or functional variants thereof, in the order
CRE0051, CRE0058,
CRE0065, CRE0066, and then the promoter element (in an upstream to downstream
direction).
[00326] The promoter element can be any suitable proximal or minimal promoter.
In some
preferred embodiments, the promoter element is a minimal promoter. Where the
promoter is a
proximal promoter, it is generally preferred that the proximal promoter is
liver-specific.
[00327] In some preferred embodiments, the promoter element is CRE0052 (also
referred to as
(I6PC) (SEQ ID NO: 126). CRE0052 is a minimal promoter (also referred to as a
core promoter).
[00328] In some embodiments, the liver-specific promoter comprises the
following regulatory
elements (or functional variants thereof): CRE0051, CRE0058, CRE0065, CRE0066
then CRE0052
(SEQ ID NO: 126).
The sequence of CRE0051 (SEQ ID NO: 97) and variants thereof are set out
above.
[00329] In some embodiments, the CRE comprising or consisting of CRE0058 (SEQ
ID NO: 113),
or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, 100 or fewer nucleotides, or 80 or fewer nucleotides.
Functional variants
thereof may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%,
or 99% identical thereto.
[00330] Functional variants of CRE0058 (SEQ ID NO: 113) are regulatory
elements with sequences
which vary from CRE0058, but which substantially retain their activity as
liver-specific CREs. It will
be appreciated by the skilled person that it is possible to vary the sequence
of a CRE while retaining
its ability to bind to the requisite TFs and enhance expression. A functional
variant can comprise
substitutions, deletions and/or insertions compared to a reference CRE,
provided they do not render
the CRE non-functional.
[00331] In some embodiments, a functional variant of CRE0058 (SEQ ID NO: 113)
can be viewed
as a CRE which, when substituted in place of CRE0058 in a promoter,
substantially retains its
activity. For example, a promoter which comprises a functional variant of
CRE0058 substituted in
place of CRE0058 preferably retains 80% of its activity, more preferably 90%
of its activity, more
preferably 95% of its activity, and yet more preferably 100% of its activity
(compared to the reference
promoter comprising CRE0058 (SEQ ID NO: 113)). For example, considering
promoter SP0265
(SEQ ID NO: 94) as an example, CRE0058 in 5P0265 can be replaced with a
functional variant of
CRE0058, and the promoter substantially retains its activity. Retention of
activity can be assessed by
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comparing expression of a suitable reporter under the control of the reference
promoter with an
otherwise identical promoter comprising the substituted CRE under equivalent
conditions.
[00332] In some embodiments it is preferred that the functional variant of
CRE0058 (SEQ ID NO:
113) comprises transcription factor binding sites (11-TBS) for the same liver-
specific transcription
factors (TF) as CRE0058. The liver-specific TFBS present in CRE0058, listed in
the order in which
they are present, are: HNF4 (SEQ ID NO: 115) and c/EBP (SEQ ID NO: 116). The
functional variant
of CRE0058 thus preferably comprises all of these TFBS. Preferably, they are
present in the same
order that they are present in CRE0058, i.e. in the order FINF4 then c/EBP.
When the CRE is
associated with a promoter and gene, this order is preferably considered in an
upstream to
downstream direction (i.e. in the direction from distal from the transcription
start site (TSS) to
proximal to the TSS). Spacer sequences may be provided between adjacent TFBS.
In some
embodiments the TFBS may suitably overlap, provided they remain functional,
i.e. overlapping
sequences are both able to bind their respective TFs..
[00333] In some embodiments, the functional variant of CRE0058 (SEQ ID NO:
113) comprises the
following TFBS sequences: CGCCCTTTGGACC (HNF4) (SEQ ID NO: 115) and
GACCTTTTGCAATCCTGG (c/EBP) (SEQ ID NO: 116), sequences complementary thereto,
or
functional variants of these TFBS sequences that maintain the ability to bind
to their respective TF..
These may be present in the same order as CRE0058, i.e. the order in which
they are set out above.
As discussed above, it is well-known in the art that there is sequence
variability associated with
TFBS, and that for a given TFBS there is typically a consensus sequence, from
which some degree of
deviation is typically present.
[00334] In some embodiments, the functional variant of CRE0058 comprises the
sequence:
GCGCCC1TIGGACCTI1TGCAATCCTGG (SEQ ID NO: 114), or a sequence that is at least
70%,
80%, 90%, 95% or 99% identical thereto.
In some embodiments, the CRE consists of SEQ ID NO: 113 or 114 or a functional
variant thereof.
[00335] It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleotide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 113 or 114 or
a fmictional
variant thereof fall within the scope of the invention. Single stranded
nucleic acids comprising the
sequence according to SEQ ID NO: 113 or 114, or a functional variant thereof,
also fall within the
scope of the invention.
[00336] In some embodiments, the CRE comprising or consisting of CRE0058, or a
functional
variant thereof, has a length of 120 or fewer nucleotides, 80 or fewer
nucleotides, 60 or fewer
nucleotides, or 40 or fewer nucleotides.
[00337] In some embodiments, the CRE comprising or consisting of CRE0065 (SEQ
ID NO: 117),
or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, 100 or fewer nucleotides, or 80 or fewer nucleotides.
Functional variants thereof
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may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or
99% identical thereto.
[00338] Functional variants of CRE0065 (SEQ ID NO: 117) are regulatory
elements with sequences
which vary from CRE0065, but which substantially retain their activity as
liver-specific CREs. It will
be appreciated by the skilled person that it is possible to vary the sequence
of a CRE while retaining
its ability to bind to the requisite TFs and enhance expression. A functional
variant can comprise
substitutions, deletions and/or insertions compared to a reference CRE,
provided they do not render
the CRE non-functional.
[00339] In some embodiments, a functional variant of CRE0065 can be viewed as
a CRE which,
when substituted in place of CRE0065 in a promoter, substantially retains its
activity. For example, a
promoter which comprises a finictional variant of CRE0065 substituted in place
of CRE0065
preferably retains 80% of its activity, more preferably 90% of its activity,
more preferably 95% of its
activity, and yet more preferably 100% of its activity (compared to the
reference promoter comprising
CRE0065). For example, considering promoter 5P0265 (SEQ ID NO: 94) as an
example, CRE0065
in SP0265 can be replaced with a functional variant of CRE0065, and the
promoter substantially
retains its activity. Retention of activity can be assessed by comparing
expression of a suitable
reporter under the control of the reference promoter with an otherwise
identical promoter comprising
the substituted CRE under equivalent conditions.
[00340] In some embodiments it is preferred that the functional variant of
CRE0065 comprises
TFBS for the same liver-specific TFs as CRE0065. The liver-specific TFBS
present in CRE0065,
listed in the order in which they are present, are: RXR Alpha (SEQ ID NO:
119), YINF3 (SEQ ID NO:
120) and FINF3 (SEQ ID NO: 121). The functional variant of CRE0065 thus
preferably comprises all
of these TFBS. Preferably, they are present in the same order that they are
present in CRE0065, i.e, in
the order RXR Alpha, FINF3 then FINF3. When the cis-regulatory element is
associated with a
promoter and gene, this order is preferably considered in an upstream to
downstream direction (i.e. in
the direction from distal from the transcription start site (TSS) to proximal
to the TSS). Spacer
sequences may be provided between adjacent TFBS. In some embodiments the TFBS
may suitably
overlap, provided they remain functional, i.e. overlapping sequences are both
able to bind their
respective TFs.
[00341] In some embodiments, the functional variant of CRE0065 comprises the
following TFBS
sequences: ACTGAACCCTTGACCCCTGCCCT (RXR Alpha) (SEQ ID NO: 119), CTGTTTGCCC
(HNF3) (SEQ ID NO: 120), and CTATTTGCCC (FINF3) (SEQ ID NO: 121), sequences
complementary thereto, or functional variants of these TFBS sequences that
maintain the ability to
bind to their respective TF. These may be present in the same order as
CRE0065, i.e. the order in
which they are set out above. As discussed above, it is well-known in the art
that there is sequence
variability associated with TFBS, and that for a given TFBS there is typically
a consensus sequence,
from which some degree of deviation is typically present.
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[00342] In some embodiments, the functional variant of CRE0065 comprises the
sequence:
[00343] ACTGAACCCTTGACCCCT-Na-CTGTTTGCCC-Nb-TA'TTTGCCC (SEQ ID NO: 118),
or a sequence that is at least 70%, 80%, 90%, 95% or 99% identical thereto,
wherein Na and Nb
represent optional spacer sequences. When present, Na optionally has a length
of from 14 to 30
nucleotides, preferably from 18 to 26 nucleotides, and more preferably 22
nucleotides. When present,
Nb optionally has a length of from 1 to 10 nucleotides, preferably from 2 to 6
nucleotides, and more
preferably 4 nucleotides. In some embodiments, the CRE consists of SEQ ID NO:
117 or 118, or a
functional variant thereof
[00344] It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleotide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 117 or 118 or
a functional
variant thereof fall within the scope of the invention. Single stranded
nucleic acids comprising the
sequence according to SEQ ID NO: 117 or 118 or a fiinctional variant thereof
also fall within the
scope of the invention.
[00345] In some preferred embodiments, the CRE comprising or consisting of
CRE0065, or a
functional variant thereof, has a length of 200 or fewer nucleotides, 150 or
fewer nucleotides, 125 or
fewer nucleotides, 90 or fewer nucleotides, or 72 or fewer nucleotides.
[00346] In some embodiments, the CRE comprising or consisting of CRE0066 (SEQ
ID NO: 122),
or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, 100 or fewer nucleotides, or 80 or fewer nucleotides
Functional variants thereof
may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or
99% identical thereto.
[00347] Functional variants of CRE0066 (SEQ ID NO: 122) are regulatory
elements with sequences
which vary from CRE0066, but which substantially retain their activity as
liver-specific CREs. It will
be appreciated by the skilled person that it is possible to vary the sequence
of a CRE while retaining
its ability to bind to the requisite TFs and enhance expression. A functional
variant can comprise
substitutions, deletions and/or insertions compared to a reference CRE,
provided they do not render
the CRE non-functional.
1003481 In some embodiments, a functional variant of CRE0066 can be viewed as
a CRE which,
when substituted in place of CRE0066 in a promoter, substantially retains its
activity. For example, a
promoter which comprises a functional variant of CRE0066 substituted in place
of CRE0066
preferably retains 80% of its activity, more preferably 90% of its activity,
more preferably 95% of its
activity, and yet more preferably 100% of its activity (compared to the
reference promoter comprising
CRE0066 (SEQ ID NO: 122). For example, considering promoter 5P0265 (SEQ ID NO:
94) as an
example, CRE0066 in 5P0265 can be replaced with a functional variant of
CRE0066, and the
promoter substantially retains its activity. Retention of activity can be
assessed by comparing
expression of a suitable reporter under the control of the reference promoter
with an otherwise
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identical promoter comprising the substituted CRE under equivalent conditions.
[00349] In some embodiments, it is preferred that the functional variant of
CRE0066 comprises
transcription factor binding sites (TFBS) for the same liver-specific
transcription factors (TF) as
CRE0066. The liver-specific TFBS present in CRE0066, listed in the order in
which they are present,
are: FINF4G (SEQ ID NO: 124) and FOS::JUN (SEQ ID NO: 125). The functional
variant of
CRE0066 thus preferably comprises all of-these TFBS. Preferably, they are
present in the same order
that they are present in CRE0066, i.e. in the order HNF4G then FOSAUN. When
the cis-regulatory
element is associated with a promoter and gene, this order is preferably
considered in an upstream to
downstream direction (i.e. in the direction from distal from the transcription
start site (TSS) to
proximal to the TSS). Spacer sequences may be provided between adjacent TFBS.
In some
embodiments the TFBS may suitably overlap, provided they remain functional,
i.e. overlapping
sequences are both able to bind their respective TFs.
[00350] In some embodiments, the functional variant of CRE0066 (SEQ ID NO:
122) comprises the
following TFBS sequences: GCAGGGCAAAGTGCA (HNF4G) (SEQ ID NO: 124) and
GATGACTCAG (FOSAUN) (SEQ ID NO: 125), sequences complementary thereto, or
functional
variants of these TFBS sequences that maintain the ability to bind to their
respective TF. These may
be present in the same order as CRE0066, i.e. the order in which they are set
out above. As discussed
above, it is well-known in the art that there is sequence variability
associated with TFBS, and that for
a given TFBS there is typically a consensus sequence, from which some degree
of deviation is
typically present.
[00351] In some embodiments, the functional variant of CRE0066 (SEQ ID NO:
122) comprises the
sequence: GCAGGGCAAAGTGCA-Na-GATGACTCAG (SEQ ID NO: 123) or a sequence that is
at
least 70%, 80%, 90%, 95% or 99% identical thereto, wherein Na represents an
optional spacer
sequence. When present, Na optionally has a length of from 10 to 28
nucleotides, preferably from 14
to 24 nucleotides, and more preferably 19 nucleotides.
In some embodiments, the CRE consists of CRE0066 or a functional variant
thereof.
[00352] It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleofide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 122 or 123 or
a functional
variant thereof fall within the scope of the invention. Single stranded
nucleic acids comprising the
sequence according to SEQ ID NO: 122 or 123, or a functional variant thereof,
also fall within the
scope of the invention.
[00353] In some preferred embodiments, the CRE comprising or consisting of
CRE0066 or a
functional variant thereof has a length of 200 or fewer nucleotides, 150 or
fewer nucleotides, 125 or
fewer nucleotides, 100 or fewer nucleotides, or 87 or fewer nucleotides.
[00354] In some embodiments, the promoter comprises the promoter element
CRE0052 (also
referred to as G6PC) (SEQ ID NO: 126) or a functional variant or functional
fragment thereof
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Functional variants thereof may have a sequence that is at least 60%, 65%,
70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
1003551 Functional variants of CRE0052 (SEQ ID NO: 126) substantially retain
the ability of
CRE0052 to act as a liver-specific promoter element. For example, when a
functional variant of
CRE0052 is substituted into liver-specific promoter 5P0265, the modified
promoter retains at least
80% of its activity, more preferably at least 90% of its activity, more
preferably at least 95% of its
activity, and yet more preferably 100% of the activity of 5P026.5.
1003561 In one embodiment the liver-specific promoter comprises SEQ ID NO: 94,
or a functional
variant thereof. In some embodiments, functional variants may have a sequence
that is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 94. The
promoter having a sequence according to SEQ ID NO: 94 is referred to as 5P0265
(also known as
SP131A1 or LVR 131_A1). A promoter comprising or consisting of SEQ ID NO: 94
is particularly
preferred in some embodiments.
1003571 In some embodiments, the liver-specific promoter is SEQ ID NO: 94 and
comprises the
following components: CRE0051 (SEQ ID NO: 97); CRE0058 (SEQ ID NO: 113);
CRE0065 (SEQ
ID NO: 117), CRE0066 (SEQ ID NO: 122), CRE0052 (SEQ ID NO: 126) ; or
functional variants of
SEQ ID NO: 97, 113, 117, 122 or 126 which may have a sequence that is at least
60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
b.SP0239 and variants thereof
1003581 In some embodiments, the promoter is a synthetic liver-specific
promoter comprising the
following CREs: CRE0018 (SEQ ID NO: 151), CRE0051 (SEQ ID NO: 97), CRE0058
(SEQ ID NO:
113), CRE0065 (SEQ ID NO: 117) and CRE0066 (SEQ ID NO: 122), or functional
variants thereof.
Typically, the CREs are operably linked to a promoter element. In some
preferred embodiments, the
liver-specific promoter comprises said CREs, or functional variants thereof,
in the order CRE0018,
CRE0051, CRE0058, CRE0065, CRE0066, and then the promoter element (in an
upstream to
downstream direction).
1003591 The promoter element can be any suitable proximal or minimal promoter.
In some
preferred embodiments the promoter element is CRE0052 (also referred to as
G6PC). CRE0052 is a
minimal promoter (also referred to as a core promoter).
1003601 In some embodiments the liver-specific promoter comprises the
following elements (or
functional variants thereof): CRE0018, CRE0051, CRE0058, CRE0065, CRE0066 and
then
CRE0052.
1003611 The sequences of CRE0051, CRE0058, CRE0065, and CRE0066 and the
promoter element
CRE0052, and functional variants thereof, are set out above.
1003621 CRE0018 has the sequence of SEQ ID NO: 151 or a functional variant or
functional
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fragment thereof. Functional variants thereof may have a sequence that is at
least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
1003631 Functional variants of CRE0018 (SEQ ID NO: 151) are regulatory
elements with sequences
which vary from CRE0018, but which substantially retain their activity as
liver-specific CREs. It will
be appreciated by the skilled person that it is possible to vary the sequence
of a CRE while retaining
its ability to bind to the requisite TFs and enhance expression. A functional
variant can comprise
substitutions, deletions and/or insertions compared to a reference CRE,
provided they do not render
the CRE substantially non-functional.
1003641 In some embodiments, a functional variant of CRE0018 can be viewed as
a CRE which,
when substituted in place of CRE0018 in a promoter, substantially retains its
activity. For example, a
promoter which comprises a finictional variant of CRE0018 substituted in place
of CRE0018
preferably retains 80% of its activity, more preferably 90% of its activity,
more preferably 95% of its
activity, and yet more preferably 100% of its activity (compared to the
reference promoter comprising
CRE0018). For example, considering promoter 5P0239 as an example, CRE0018 in
5P0239 in can
be replaced with a functional variant of CRE0018, and the promoter
substantially retains its activity.
Retention of activity can be assessed by comparing expression of a suitable
reporter under the control
of the reference promoter with an otherwise identical promoter comprising the
substituted CRE under
equivalent conditions.
1003651 In some embodiments, the functional variant of CRE0018 comprises TFBS
for the same
liver-specific TFs as CRE0018. The liver-specific TFBS present in CRE0018,
listed in the order in
which they are present, are: IRF (SEQ ID NO: 129), NF1 (SEQ ID NO: 130), HNF3
(SEQ ID NO:
131), HBLF (SEQ ID NO: 132), RXRa (SEQ ID NO: 133), EF-C (SEQ ID NO: 134), NF1
(SEQ ID
NO: 135), and c/EBP (SEQ ID NO: 136). The functional variant of CRE0018 thus
preferably
comprises all of these TFBS. Preferably, they are present in the same order
that they are present in
CRE0018, i.e. in the order IRF, NF1, HNF3, HBLF, RXRa, EF-C, NF1, and then
c/EBP. When the
CRE is associated with a promoter and gene, this order is preferably
considered in an upstream to
downstream direction (i.e. in the direction from distal from the transcription
start site (TSS) to
proximal to the TSS). Spacer sequences may be provided between adjacent TFBS.
In some
embodiments the TFBS may suitably overlap, provided they remain functional,
i.e. overlapping
sequences are both able to bind their respective TFs.
1003661 In some embodiments the functional variant of CRE0018 comprises the
following TFBS
sequences: CTTTCACTTTC (IRF) (SEQ ID NO: 129), TCGCCAA (NF1) (SEQ ID NO: 130),
TGTGTAAACA (HNF3) (SEQ ID NO: 131), TGTAAACAATA (FIBLF) (SEQ ID NO: 132),
CTGAACCTTTACCC (RXRa) (SEQ ID NO: 133), GTTGCCCGGCAAC (EF-C) (SEQ ID NO:
134), CAGGTCTGTGCCAAG (NF1) (SEQ ID NO: 135), TGCCAAGTGTTTG (c/EBP) (SEQ ID
NO: 136), sequences complementary thereto, or functional variants of these
TFBS sequences that
maintain the ability to bind to their respective TF of SEQ ID NO: 129-136.
These may be present in
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the same order as CRE0018, i.e. the order in which they are set out above. As
discussed above, it is
well-known in the art that there is sequence variability associated with TFBS,
and that for a given
TFBS there is typically a consensus sequence, from which some degree of
deviation is typically
present.
1003671 In some embodiments of the invention, the functional variant of
CRE0018 comprises the
sequence: CTITCACITTCTCGCCAA-Na-TGTGTAAACAATA-Nb-CTGAACCTITACCC-Nc-
GTTGCCCGGCAAC-Nd-CAGGTCTGTGCCAAGTGITTG (SEQ ID NO: 128), or a sequence that
is at least 70%, 80%, 90%, 95% or 99% identical thereto, wherein Na, Nb, Nc,
and Nd represent
optional spacer sequences. When present, Na optionally has a length of from 10
to 20 nucleotides,
preferably from 13 to 17 nucleotides, and more preferably 15 nucleotides. When
present, Nb
optionally has a length of from 1 to 10 nucleotides, preferably from 1 to 5
nucleotides, more
preferably 1 nucleotide. When present, Nc optionally has a length of from 1 to
10 nucleotides,
preferably 1 to 5 nucleotides, and more preferably 1 nucleotide. When present,
Nd suitably has a
length of from 1 to 10 nucleotides, preferably from 2 to 8 nucleotides in
length, and more preferably 3
nucleotides in length.
1003681 In some embodiments of the invention the CRE consists of SEQ ID NO:
127 or 128 or a
functional variant thereof.
1003691 It will be noted that the CRE or functional variant thereof can be
provided on either strand
of a double stranded polynucleotide and can be provided in either orientation.
As such,
complementary and reverse complementary sequences of SEQ ID NO: 128 or 129 or
a functional
variant thereof fall within the scope of the invention. Single stranded
nucleic acids comprising the
sequence according to SEQ ID NOS: 128 or 129 or a fiuictional variant thereof
also fall within the
scope of the invention.
1003701 In some embodiments, the CRE comprising or consisting of CRE0018 (SEQ
ID NO: 151),
or a functional variant thereof, has a length of 200 or fewer nucleotides, 150
or fewer nucleotides, 125
or fewer nucleotides, or 103 or fewer nucleotides.
1003711 In one embodiment the liver-specific promoter comprises or consist of:
SEQ ID NO: 93, or
a functional variant thereof. The promoter having a sequence according to SEQ
ID NO: 93 is referred
to as SP0239. Functional variants of SP0239 can have a sequence that is at
least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
1003721 Accordingly, in some embodiments, the liver-specific promoter is
5P0239 (SEQ ID NO:
93) and comprises the following components: CRE0018 (SEQ ID NO: 151), CRE0051
(SEQ ID NO:
97), CRE0058 (SEQ ID NO: 113), CRE0065 (SEQ ID NO: 117) and CRE0066 (SEQ ID
NO: 122,
and CRE0052 (SEQ ID NO: 126); or functional variants may have a sequence that
is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
c. SP0240 and variants thereof
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1003731 In some embodiments, the promoter is a synthetic liver-specific
promoter comprising
CRE0018 operably linked to a promoter element. In some preferred embodiments,
the liver-specific
promoter comprises CRE0018, or immediately upstream of the promoter element.
1003741 The promoter element can be any suitable proximal or minimal promoter.
In some
preferred embodiments the promoter element is CRE0006 (SEQ ID NO: 137).
CRE0006 is a liver-
specific proximal promoter.
1003751 In some embodiments the liver-specific promoter comprises the
following elements (or
functional variants thereof): CRE0018 and then CRE0006.
1003761 The sequence of CRE0018 and variants thereof are set out above.
1003771 CRE0006 is a proximal promoter and comprises TFBS for liver-specific
TFs upstream of
the TSS. The liver-specific TFBS present in CRE0006, listed in order, are HNF4
(SEQ ID NO: 138),
RXRa (SEQ ID NO: 139), HNF4 (SEQ ID NO: 140), c/EBP (SEQ ID NO: 141), and
IINF3 (SEQ ID
NO: 142), and optionally pl@VTN (SEQ ID NO: 143). The functional variant of
CRE0006 thus
preferably comprises these THIS. Preferably, they are present in the same
order that they are present
in CRE0006, i.e. in the order FINF4, c/EBP, HNF3, and HNF3. In some
embodiments the TFBS
overlap, provided they remain functional, i.e. overlapping sequences are both
able to bind their
respective TFs.
1003781 pl@VTN (SEQ ID NO: 143), represents the transcription start site (TSS)
in CRE0006, as
determined by Cap Analysis of Gene Expression (CAGE).
1003791 In some embodiments, a functional variant of CRE0006 comprises a
sequence which is at
least 70% identical to SEQ ID NO: 137 (preferably at least 80%, 90%, 95% or
99% identical to SEQ
ID NO: 25), which contains TFBS for HNF4, RXRa, HNF4, c/EBP, and YINF3, and
preferably which
contains a TSS sequence which is at least 80%, 90%, 95% or completely
identical to THIS for HNF4,
RXRa, HNF4, c/EBP, and EINF3 downstream of said TFBS.
1003801 In some embodiments, a functional variant of CRE0006 comprises a
sequence which has at
least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO: 137, and which further
comprises the
following TFBS: HNF4 (SEQ ID NO: 138) at or near position 25-37; RXRa (SEQ ID
NO: 139) at or
near position 73-83; HNF4 (SEQ ID NO: 140) at or near position 74-86; c/EBP
(SEQ ID NO: 141) at
or near position 123-136; arid HNF3 (SEQ ID NO: 142) at or near position 129-
137; and which
comprises a TSS sequence which is at least 80%, 90%, 95% or completely
identical to SEQ ID NO:
143 at or near position 166-196, positions being numbered with reference to
SEQ ID NO: 137. At or
near in the present context suitably means within 10, 5, 4, 3, 2, or 1
nucleotide of the recited position
with reference to SEQ ID NO: 137. Suitable TFBS sequences are SEQ ID Nos: 138-
142, but
alternative TFBS sequences can be used.
1003811 In one embodiment the liver-specific promoter comprises or consist of
SEQ ID NO: 95, or a
functional variant thereof The promoter having a sequence according to SEQ ID
NO: 95 is referred
to as 5P0240. Functional variants of SP0240 can have a sequence that is at
least 60%, 65%, 70%,
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75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
d. SP0246 and variants thereof
[00382] In some embodiments, the promoter is a synthetic liver-specific
promoter comprising the
following CREs: CRE0051, CRE0058, and CRE0065, or functional variants thereof
Typically, the
CREs are operably linked to a promoter element. In some preferred embodiments,
the liver-specific
promoter comprises said CREs, or functional variants thereof, in the order
CRE0051, CRE0058, and
CRE0065, and then the promoter element (in an upstream to downstream
direction).
[00383] The promoter element can be any suitable proximal or minimal promoter.
In some
preferred embodiments the promoter element is CRE0052 (also referred to as
G6PC). CRE0052 is a
minimal promoter (also referred to as a core promoter).
[00384] In some embodiments the liver-specific promoter comprises the
following elements (or
functional variants thereof): CRE0051, CRE0058, CRE0065, and then CRE0052. The
sequences of
CRE0051, CRE0058, CRE0065 and the promoter element CRE0052, and functional
variants thereof,
are set out above.
[00385] In one embodiment the liver-specific promoter comprises or consist of
SEQ ID NO; 96, or a
functional variant thereof. The promoter having a sequence according to SEQ ID
NO: 96 is referred
to as SP0246. Functional variants of SP0246 can have a sequence that is at
least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 96.
e. SP0131 and variants thereof
[00386] In some embodiments, the promoter is a synthetic liver-specific
promoter comprising the
following CREs: CRE0058, CRE0065 and CRE0066, or functional variants thereof
Typically, the
CREs are operably linked to a promoter element. In some preferred embodiments,
the liver-specific
promoter comprises said CREs, or functional variants thereof, in the order
CRE0058, CRE0065,
CRE0066 and then the promoter element (in an upstream to downstream
direction).
[00387] The promoter element can be any suitable proximal or minimal promoter.
In some
preferred embodiments the promoter element is CRE0052 (also referred to as
(16PC). CRE0052 is a
minimal promoter (also referred to as a core promoter).
[00388] The sequences of CRE0058, CRE0065, and CRE0066 and the promoter
element CRE0052,
and functional variants thereof, are set out above.
[00389] SEQ ID NO: 141, or a ftmctional variant thereof The promoter having a
sequence
according to SEQ ID NO: 141 is referred to as SP0131. Functional variants of
SP0131 can have a
sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99%
identical thereto.
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t Composite Promoters:
[00390] In some embodiments, the liver-specific promoters as set out above are
operably linked to
one or mom additional regulatory sequences. An additional regulatory sequence
can, for example,
enhance expression compared to the liver-specific promoter which is not
operably linked the
additional regulatory sequence. Generally, it is preferred that the additional
regulatory sequence does
not substantively reduce the specificity of the liver-specific promoter.
[00391] For example, the liver-specific promoter can be operably linked to a
sequence encoding a
UTR (e.g. a 5' and/or 3' UTR), an intron, or such.
[00392] In some embodiments, the liver-specific promoter is operably linked to
sequence encoding
a UTR, e.g. a 5' UTR. A 5' UTR can contain various elements that can regulate
gene expression. The
5' UTR in a natural gene begins at the transcription start site and ends one
nucleotide before the start
codon of the coding region. It should be noted that 5' UTRs as referred to
herein may be an entire
naturally occurring 5' UTR or it may be a portion of a naturally occurring 5'
UTR.. The 5'UTR can
also be partially or entirely synthetic. In eukaryotes, 5' UTRs have a median
length of approximately
150 nt, but in some cases they can be considerably longer. Regulatory
sequences that can be found in
5' UTRs include, but are not limited to:
Binding sites for proteins, that may affect the mRNA's stability or
translation;
Riboswitches;
Sequences that promote or inhibit translation initiation; and
Introns within 5' UTRs have been linked to regulation of gene expression and
mRNA export.
[00393] In some embodiments, a liver-specific promoter as set out above is
operably linked to a
sequence encoding a 5' UTR derived from the CMV major immediate gene (CMV-IE
gene). For
example, the 5' UTR from the CMV-IE gene suitably comprises the CMV-IE gene
exon 1 and the
CMV-IE gene exon 1, or portions thereof In some cases, the promoter element
may be modified in
view of the linkage to the 5 4UTR, for example sequences downstream of the
transcription start site
(TSS) in the promoter element can be removed (e.g. replaced with the 5' UTR).
[00394] The CMV-IE 5 'UTR is described in Sitnari, et al., Molecular Medicine
4: 700-706, 1998
"Requirements for Enhanced Transgene Expression by Untratzslated Sequences
from the Human
Cytomegalovirus Immediate-Early Gene", which is incorporated herein by
reference. Variants of the
CMV-IE 5' UTR sequences discussed in Simari, et al. are also set out in
W02002/031137,
incorporated by reference, and the regulatory sequences disclosed therein can
also be used. Other
UTRs that can be used in combination with a promoter are known in the art,
e.g. in Leppek, K., Das,
R. & Barna, M. "Functional 5' UTR mRNA structures in eukwyotic translation
regulation and how to
find them". Nat Rev Mol Cell Biol 19, 158-174 (2018), incorporated by
reference.
[00395] In some embodiments the sequence encoding the 5' UTR comprises SEQ ID
NO: 145, or a
functional variant thereof In some embodiments, functional variants may have a
sequence that is at
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least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
thereto. SEQ
ID NO: 145 encodes a CMV-IE 5' UTR.
1003961 In some embodiments the 5' UTR comprises a nucleic acid motif that
functions as the
protein translation initiation site, e.g. sequences that define a Kozak
sequence in the mRNA produced.
For example, in some embodiments, the sequence encoding the 5' UTR comprises
the sequence motif
GCCACC (SEQ ID NO: 153) at or near its 3' end. Other Kozak sequences or other
protein
translation initiation sites can be used, as is known in the art (e.g. Marilyn
Kozak, "Point Mutations
Defme a Sequence Flanking the AUG Initiator Ctxlon That Modulates Translation
by Eukaryotic
Ribosomes" Cell, Vol, 44, 283-292, January 31, 1986; Marilyn Kozak "At Least
Six Nucleotides
Preceding the AUG Initiator Codon Enhance Translation in Mammalian Cells" J.
Mol. Rid. (1987)
196,947-950; Marilyn Kozak "An analysis of 5'-noncoding sequences from 699
vertebrate messenger
RNAs" Nucleic Acids Research. Vol. 15 (20) 1987, all of which are incorporated
herein by
reference). The protein translation initiation site (e.g. Kozak sequence) is
preferably positioned
immediately adjacent to the start codon.
1003971 In some embodiments the sequence encoding the 5' UTR comprises SEQ ID
NO: 438, or a
functional variant thereof In some embodiments, functional variants may have a
sequence that is at
least 60%, 65%, 70%, 75%, 80%, 85%, 900/c, 95%, 96%, 97%, 98%, or 99%
identical thereto. This 5'
UTR comprises six nucleotides of SEQ ID NO: 153 which define a Kozak sequence
at the 3' end of
the CMV-IE 5' UTR.
1003981 In some embodiments, the SP0412 promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter/5' UTR
regulatory
construct. Herein, such composite promoter/5' UTR constructs may be referred
to simply as
"composite promoters", or in some cases simply "promoters" for brevity.
[00399] In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 92, or
a functional variant thereof. In some embodiments, finictional variants may
have a sequence that is at
least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO:
92.
[00400] This composite promoter comprises 5P0412 operably linked to a sequence
encoding the 5'
UTR from the CMV-IE gene (SEQ ID NO: 145) and the GCCACC (SEQ ID NO: 153)
Kozak
sequence discussed above. This (composite) promoter is referred to as 5P0422
(SEQ ID NO: 92).
SP0422 is a preferred liver specific promoter in some embodiments. As
discussed above, the 5' UTR
suitably comprises a nucleic acid motif that functions as the protein
translation initiation site, e.g.
sequences that define a Kozak sequence. In the sequence above, the 5' UTR
comprises the sequence
motif GCCACC (SEQ ID NO: 153) at its 3' end, but this sequence motif can be
omitted or alternative
sequences can be used.
[00401] In some embodiments, the 5P0265 promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter (5P0236-
5UTR).
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1004021 In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 146,
or a functional variant thereof. In some embodiments, functional variants may
have a sequence that is
at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID
NO: 146.
1004031 This composite promoter comprises SP0265(SEQ ID NO: 94) operably
linked to the 5'
UTR from the CMV-IE gene (SEQ ID NO: 145) and the GCCACC (SEQ ID NO: 153)
Kozak
sequence. This (composite) promoter is referred to as 5P0420. In this
promoter, a short sequence
downstream of the TSS in the CRE0052 promoter element have been replaced with
sequences from
the 5' UTR from the CMV-IE. Thus, this promoter actually comprises a minor
variant of 5P0265
with a modification to CRE0052 whereby some sequence has been removed. SP0420
is preferred in
some embodiments. As discussed above, the 5' UTR suitably comprises a nucleic
acid motif that
functions as the protein translation initiation site, e.g. sequences that
define a Kozak sequence. In the
sequence above, the 5' UTR comprises the sequence motif GCCACC (SEQ ID NO:
153) at its 3' end,
but this sequence motif can be omitted or alternative sequences can be used.
1004041 In some embodiments, the 5P0239 promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter (5P0239-
UTR).
1004051 In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 147,
or a functional variant thereof. In some embodiments, functional variants may
have a sequence that is
at least 60%, 65%, 70%, 75%, 80%s 85%, 90%, 95%, 96%s 97%, 98%, or 99%
identical to SEQ ID
NO: 147.
1004061 This composite promoter/5' UTR construct comprises 5P0239 operably
linked to the 5'
UTR from the CMV-IE gene and the GCCACC (SEQ ID NO: 153) Kozak sequence. This
(composite) promoter is referred to as SP0421. Again, in this promoter, a
short sequence downstream
of the TSS in the CRE0052 promoter element have been replaced with sequences
from the 5' UTR
from the CMV-IE. Thus, this promoter actually comprises a minor variant of
SP0239 with a
modification to CRE0052 whereby some sequence has been removed. SP0421 is
preferred in some
embodiments. As discussed above, the 5' UTR suitably comprises a nucleic acid
motif that functions
as the protein translation initiation site, e.g. sequences that define a Kozak
sequence. In the sequence
above, the 5' UTR comprises the sequence motif GCCACC (SEQ ID NO: 153) at its
3' end, but this
sequence motif can be omitted or alternative sequences can be used.
1004071 In some embodiments, the SP0240 promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter.
1004081 In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 148,
or a functional variant thereof In some embodiments, functional variants may
have a sequence that is
at least 60%, 65%, 70%, 75%, 800/n, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical thereto.
1004091 This composite promoter/5' UTR construct comprises 5P0240 operably
linked to the 5'
UTR from the CMV-IE gene and the GCCACC (SEQ ID NO: 153) Kozak sequence. This
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(composite) promoter is referred to as SP0240-UTR. Again, in this promoter, a
short sequence
downstream of the TSS in the CRE0006 promoter element have been replaced with
sequences from
the 5' UTR from the CMV-IE. Thus, this promoter actually comprises a minor
variant of 5P0240
with a modification to CRE0006 whereby some sequence has been removed. SP0240-
UTR is
preferred in some embodiments. As discussed above, the 5' UTR suitably
comprises a nucleic acid
motif that functions as the protein translation initiation site, e.g.
sequences that defme a Kozak
sequence. In the sequence above, the 5' UTR comprises the sequence motif
(JCCACC (SEQ ID NO:
153) at its 3' end, but this sequence motif can be omitted or alternative
sequences can be used.
1004101 In some embodiments, the 5P0246 promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter.
1004111 In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 149
(SP0246-UTR), or a functional variant thereof. In some embodiments, functional
variants may have a
sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99%
identical thereto.
1004121 This composite promoter/5' UTR construct comprises 5P0246 operably
linked to the 5'
UTR from the CMV-IE gene and the GCCACC (SEQ ID NO: 153) Kozak sequence. This
(composite) promoter is referred to as SP0246-UTR. Again, in this promoter, a
short sequence
downstream of the TSS in the CRE0052 promoter element have been replaced with
sequences from
the 5' UTR from the CMV-IE. Thus, this promoter actually comprises a minor
variant of SP0246
with a modification to CRE0052 whereby some sequence has been removed. SP0246-
UTR is
preferred in some embodiments. As discussed above, the 5' UTR suitably
comprises a nucleic acid
motif that functions as the protein translation initiation site, e.g.
sequences that define a Kozak
sequence. In the sequence above, the 5' UTR comprises the sequence motif
GCCACC (SEQ ID NO:
153) at its 3' end, but this sequence motif can be omitted or alternative
sequences can be used.
1004131 In some embodiments, the SP0131 Al promoter, or variants thereof, as
discussed above is
linked to a sequence encoding a 5' UTR to provide a composite promoter.
1004141 In some embodiments, the composite promoter comprises or consists of
SEQ ID NO: 150
(SP0131 Al-UTR), or a fitnctional variant thereof, In some embodiments,
functional variants may
have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or
99% identical thereto.
1004151 This composite promoter/5' UTR construct comprises SP0131 operably
linked to the 5'
UTR from the CMV-IE gene and the GCCACC (SEQ ID NO: 153) Kozak sequence. This
(composite) promoter is referred to as SP0131-UTR. Again, in this promoter, a
short sequence
downstream of the TSS in the CRE0052 promoter element have been replaced with
sequences from
the 5' UTR from the CMV-IE. Thus, this promoter actually comprises a minor
variant of SP0131
with a modification to CRE0052 whereby some sequence has been removed. SP0131-
UTR is
preferred in some embodiments. As discussed above, the 5' UM suitably
comprises a nucleic acid
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motif that functions as the protein translation initiation site, e.g.
sequences that defme a Kozak
sequence. In the sequence above, the 5' UTR comprises the sequence motif
GCCACC (SEQ ID NO:
153) at its 3' end, but this sequence motif can be omitted or alternative
sequences can be used.
1004161 In some embodiments, the liver-specific promoter is 5P0412 (SEQ ID NO:
91) and
comprises the following components: CRE0051 (SEQ ID NO: 97), CRE0067 (SEQ ID
NO: 152),
CRE0059 (SEQ ID NO: 110) and a Kozak sequence (SEQ ID NO: 153); or functional
variants may
have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or
99% identical thereto.
1004171 In some embodiments, the liver-specific promoter is 5130422 (SEQ ID
NO: 9) and
comprises the following components: CRE0051 (SEQ ID NO: 97), CRE0067 (SEQ ID
NO: 152),
CRE0059 (SEQ ID NO: 110), CMV-IE 5'UTR (SEQ ID NO: 153) and a Kozak sequence
(SEQ ID
NO: 153, or functional variants may have a sequence that is at least 60%, 65%,
70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
(iii) Functional variants of the synthetic liver-specific promoters
1004181 In some embodiments, a functional variant of a liver-specific promoter
can be viewed as a
promoter element which, when substituted in place of a reference promoter
element in a promoter,
substantially retains its activity. For example, a functional variant of liver-
specific promoter which
comprises a functional variant of a given promoter in Table 4 herein, or any
promoter listed from SEQ
ID NOS: 86 (CRM 0412), SEQ ID NO: 91 (SP0412) or SEQ ID NO: 92 (5P0422), SEQ
ID NOS: 93
(SP0239), SEQ ID NO: 94 (5P0265, also referred to SP131_Al), SEQ ID NO: 95
(SP0240) or SEQ
ID NO: 96 (5P0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-
UTR), SEQ ID
NO: 148 (5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (SP0131-A1-
UTR),
or a functional fragment or variant thereof, or any LSP selected from SEQ ID
NO: 270-341 or 342-
430, or a functional fragment or variant thereof where a functional variant or
functional fragments
preferably retains at least 35%, or at least 40%, or at least 45%, or at least
50%, or at least 55%, or at
least 60%, or at least 70% or at least 80% of its activity, more preferably at
least 90% of its activity,
more preferably at least 95% of the activity of the unchanged promoter, and
yet more preferably
100% of the activity (as compared to the unchanged promoter sequence
comprising the unmodified
promoter element). Suitable assays for assessing liver-specific promoter
activity are disclosed herein,
e.g. in Examples 12 and 13.
1004191 In some embodiments, a functional variant or a functional fragment of
a liver-specific
promoter disclosed in Table 4 herein, or any promoter listed from SEQ ID NOS:
86 (CRM 0412),
SEQ ID NO: 91 (SP0412) or SEQ ID NO: 92 (5P0422), SEQ ID NOS: 93 (5P0239), SEQ
ID NO: 94
(5130265, also referred to SP131 Al), SEQ ID NO: 95 (5P0240) or SEQ ID NO: 96
(5P0246), or
SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID NO: 148
(5P0240-
UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (SP0131-Al-UTR), or any
LSP selected
from SEQ ID NO: 270-341 or 342-430, has at least about 75% sequence identity
to, or at least about
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80% sequence identity to, at least about 90% sequence identity to, at least
about 95% sequence
identity to, at least about 98% sequence identity to the original unmodified
sequence, and also at least
35% of the promoter activity, or at least about 45% of the promoter activity,
or at least about 50% of
the promoter activity, or at least about 60% of the promoter activity, or at
least about 75% of the
promoter activity, or at least about 80% of the promoter activity, or at least
about 85% of the promoter
activity, or at least about 90% of the promoter activity, or at least about
95% of the promoter activity
of the corresponding unmodified promoter sequence.
1004201 For example, a functional variant or a functional fragment of SEQ ID
NO: 258 (5P0412) or
SEQ ID NO: 92 (5P0422) has at least about 75% sequence identity to SEQ ID NO:
91(SP0412) or
SEQ ID NO: 92(SP0422), or at least about 80% sequence identity to SEQ ID NO:
91 (SP0412) or
SEQ ID NO: 92 (5P0422), at least about 90% sequence identity to SEQ ID NO: 91
(5P0412) or SEQ
ID NO: 92 (SP0422), at least about 95% sequence identity to SEQ ID NO: 91
(SP0412) or SEQ ID
NO: 92 (5P0422), at least about 98% sequence identity to SEQ ID NO: SEQ ID NO:
91 (5P0412) or
SEQ ID NO: 92(5P0422), or the original unmodified sequence, and also at least
35% of the promoter
activity, or at least about 45% of the promoter activity, or at least about
50% of the promoter activity,
or at least about 60% of the promoter activity, or at least about 75% of the
promoter activity, or at
least about 80% of the promoter activity, or at least about 85% of the
promoter activity, or at least
about 90% of the promoter activity, or at least about 95% of the promoter
activity of the
corresponding unmodified promoter sequence of SEQ ID NO: 91 (5P0412) or SEQ ID
NO: 92
(5P0422), respectively.
1004211In some embodiments, a functional variant of a liver-specific promoter
disclosed herein
retains a significant level of sequence identity to the unmodified promoter
sequence. Suitably
functional variants comprise a sequence that is at least 60% identical to the
unmodified promoter
sequence, more preferably at least 70%, 80%, 90%, 95% or 99% identical to the
unmodified liver-
specific promoter sequence.
1004221In some embodiments, a functional fragment of a liver-specific promoter
disclosed herein
retains a significant level of sequence identity to the unmodified promoter
sequence. Suitable
fmictional fragments comprise a sequence that is at least 60% identical to the
unmodified promoter
sequence, more preferably at least 70%, 80%, 90%, 95% or 99% identical to the
unmodified liver-
specific promoter sequence.
1004231In some embodiments, a functional variant of a promoter element can be
viewed as a
promoter element which, when substituted in place of a reference promoter
element in a promoter,
substantially retains its activity. For example, a liver-specific promoter
which comprises a functional
variant of a given promoter element preferably retains at least 80% of its
activity, more preferably at
least 90% of its activity, more preferably at least 95% of its activity, and
yet more preferably 100% of
its activity (compared to the reference promoter comprising the unmodified
promoter element).
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Suitable assays for assessing liver-specific promoter activity are disclosed
herein, e.g. in Examples 12
and 13.
[00424] It should be noted that the sequences of a liver-specific promoter as
disclosed herein in Table
4, or any LSP selected from SEQ ID NO: 270-341 or 342-430 can be altered
without causing a
substantial loss of activity. Thus, functional variants of a liver-specific
promoter are discussed below
can be prepared by modifying the sequence of a liver-specific promoter
disclosed in Table 4 herein, or
any or any LSP selected from SEQ ID NO: 270-341 or 342-430, provided that
modifications which
are significantly detrimental to activity of the liver-specific promoter are
avoided. In view of the
information provided in the present disclosure, modification of a liver-
specific promoter disclosed
herein in Table 4, or any LSP selected from SEQ ID NO: 270-341 or 342-430 to
provide functional
variants is straightforward. Moreover, the present disclosure provides
methodologies for simply
assessing the functionality of any given liver-specific promoter variant
Functional variants for each
liver-specific promoter are discussed below.
1004251ln some embodiments of the invention the synthetic liver-specific
promoter comprises a
sequence from the group consisting of any promoter listed from SEQ ID NOS: 86
(CRIV1 0412), SEQ
ID NO: 91 (SP0412) or SEQ ID NO: 92 (5P0422), SEQ ID NOS: 93 (5P0239), SEQ ID
NO: 94
(SP0265, also referred to SP131_Al), SEQ ID NO: 95 (5P0240) or SEQ ID NO: 96
(SP0246), or
SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID NO: 148
(5P0240-
UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (SP0131-Al-UTR), or any
LSP selected
from SEQ ID NO: 270-341 or 342-430, or a functional variant of any thereof.
Suitably the functional
variant of any of said liver-specific promoter comprises a sequence that is at
least 70% identical to the
reference synthetic liver-specific promoter, more preferably at least 80%,
90%, 95% or 99% identical
to the reference synthetic liver-specific promoter.
1004261ln some embodiments, the functional variant thereof may suitably
comprise a sequence that is
at least 60%, 70%, 80%, 90%, 95% or 99% identical to any one of the sequences
listed in Table 4.
Additionally or alternatively, a functional variant of any one of the
sequences listed in Table 4,
suitably comprises a sequence which hybridizes under stringent conditions to
the reference sequence.
Functional variants of any one of the sequences listed in Table 4 include
variants in which one or
more of the sequence provided therein has been replaced with a functional
variant thereof as defined
above, and/or where the order of the sequences provided therein has been
altered.
1004271 In some embodiments, a functional variant of any one of the liver-
specific promoter
sequences listed in Table 4 can be viewed as a liver-specific promoter, when
at least one or more
nucleotides are substituted and it substantially retains its activity. For
example, a liver-specific
promoter which comprises a functional variant of any one of the liver-specific
promoter sequences
listed in Table 4 preferably retains 80% of its activity, more preferably 90%
of its activity, more
preferably 95% of its activity, and yet more preferably 100% of its activity
(compared to the reference
promoter sequence). For example, if a LSP comprises a nucleic acid sequence
comprising 5P0412
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(SEQ ID NO: 91) as an example, a portion of nucleotides in SP0412 (e.g., SEQ
ID NO:91) in can be
replaced with a functional variant of thereof, and the liver specific SP0412
promoter substantially
retains its activity. Retention of activity can be assessed by comparing
expression of a suitable
reporter under the control of the reference promoter with an otherwise
identical promoter comprising
the substituted nucleic acids under equivalent conditions. Suitable assays for
assessing liver-specific
promoter activity are disclosed herein, e.g. in examples 12 and 13.
1004281In some embodiments of the compositions and methods disclosed herein, a
synthetic liver-
specific promoter disclosed herein in Table 4, or any promoter listed from SEQ
ID NOS: 86 (CRM
0412), SEQ ID NO: 91 (5P0412) or SEQ ID NO: 92 (SP0422), SEQ ID NOS: 93
(5P0239), SEQ ID
NO: 94 (SP0265, also referred to SP131_A1), SEQ ID NO: 95 (5P0240) or SEQ ID
NO: 96
(SP0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID
NO: 148
(5P0240-UTR), SEQ ID NO: 149 (SP0246-UTR) or SEQ ID NO: 150 (SP0131-A1-UTR),
or any
LSP selected from SEQ ID NO: 270-341 or 342-4301s a functional variant thereof
that has length of
700, 600, 500, 450, 400, 350, 300, 250 or 200 or 150 or fewer nucleotides.
1004291 In some embodiments of the compositions and methods disclosed herein,
the synthetic liver-
specific promoter disclosed herein in Table 4, any promoter listed from SEQ ID
NOS: 86 (CRM
0412), SEQ ID NO: 91 (SP0412) or SEQ ID NO: 92 (5P0422), SEQ ID NOS: 93
(5P0239), SEQ ID
NO: 94 (5P0265, also referred to 5P131 Al), SEQ ID NO: 95 (5P0240) or SEQ ID
NO: 96
(5P0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID
NO: 148
(5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (SP0131-Al-UTR),
or any
LSP selected from SEQ ID NO: 270-341 or 342-430, comprises a synthetic liver-
specific cis-
regulatory element (CRE) or cis-regulatory module (CRM) operably linked to a
minimal promoter.
Examples of suitable minimal promoters for use in the present invention
include, but are not limited
to, the CMV-minimal promoter, MinTk minimal promoter, and the LVR_CRE0052_G6PC
minimal
promoter (SEQ ID NO: 126). In particular embodiments, the minimal promoter is
the CMV-IE
promoter comprising the sequence of SEQ ID NO: 145, a sequence that is at
least 60%, 70%, 80%,
90%, 95% or 99% identical to SEQ ID NO: 145 Exemplary promoters comprising a
CMV-IE for use
in the methods and compositions disclosed herein can be selected from, but are
not limited to, SEQ ID
NO: 92 (5P0422), SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ
ID NO:
148 (5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (SP0131-Al-
UTR).
1004301 In some embodiments of the compositions and methods disclosed herein,
a synthetic liver-
specific promoter disclosed herein in Table 4, e.g., any promoter listed from
SEQ ID NOS: 86 (CRM
0412), SEQ ID NO: 91 (5P0412) or SEQ ID NO: 92 (SP0422), SEQ ID NOS: 93
(5P0239), SEQ ID
NO: 94 (SP0265, also referred to SP131_Al), SEQ ID NO: 95 (5P0240) or SEQ ID
NO: 96
(5P0246), or SEQ ID NO: 146 (5P0265-UTR), SEQ ID NO: 147 (5P0239-UTR), SEQ ID
NO: 148
(5P0240-UTR), SEQ ID NO: 149 (5P0246-UTR) or SEQ ID NO: 150 (5P0131-Al-UTR),
or any
LSP selected from SEQ ID NO: 270-341 or 342-430 is able to increase expression
of gene in the liver
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of a subject or in a liver cell by at least 20%, at least 40%, at least 60%,
at least 80%, at least 100%, at
least 200%, at least 300%, at least 500%, at least 1000% or more relative to
the LP-1 promoter (SEQ
ID NO: 432),
1004311 In some embodiments of the compositions and methods disclosed herein,
the synthetic liver-
specific promoter disclosed herein in Table 4,or any LSP promoter selected
from SEQ ID NOS: 86,
91-96, 146-150, or any LSP selected from SEQ ID NO: 270-341 or 342-430, a
synthetic liver-specific
promoter is able to promote liver-specific transgene expression and has an
activity in liver cells which
is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%,
175%, 200%,
250%, 300%, 350% or 400% of the activity of the TTR promoter (SEQ ID NO: 431),
1004321In some embodiments of the compositions and methods disclosed herein,
the synthetic liver-
specific promoter disclosed herein in Table 4, or any LSP promoter selected
from SEQ ID NOS: 86,
91-96, 146-150, or any LSP selected from SEQ ID NO: 270-341 or 342-430, a
synthetic liver-specific
promoter is able to promote liver-specific transgene expression and has an
activity in liver cells which
is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%,
175%, 200%,
250%, 300%, 350% 01 400% of the activity of the TBG promoter (SEQ ID NO: 435).
G. Intron sequence
1004331In some embodiments, the rAAV genotype comprises an intron sequence
located 3' of the
promoter sequence and 5' of the secretory signal peptide. Intron sequences
serve to increase one or
more of: mRNA stability, mRNA transport out of nucleus and/or expression
and/or regulation of the
expressed GAA fusion polypeptide (e.g., SS-GAA fusion polypeptide or SS-IGF2-
GAA polypeptide).
In alternative embodiments, a rAAV genotype does not comprise an intron
sequence.
[00434] In some embodiments, the intron sequence is a MVM intron sequence, for
example, but not
limited to and intron sequence of SEQ ID NO: 13 or nucleic acid sequence
having at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
10043511n some embodiments, the intron sequence is a HEB2 intron sequence, for
example, but not
limited to and intron sequence of SEQ ID NO: 14 or nucleic acid sequence
having at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
10043611n some embodiments of the methods and compositions disclosed herein, a
recombinant AAV
vector comprises a heterologous nucleic acid sequence that further comprises
an intron sequence
located 5' of the sequence encoding the secretory signal peptide, and 3' of
the promoter. In some
embodiments, the intron sequence comprises a MVM sequence or a FIBB2 sequence,
wherein the
MVM sequence comprises the nucleic acid sequence of SEQ ID NO: 13, or a
nucleic acid sequence at
least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to SEQ ID NO:
13, and the HBB2 sequence comprises the nucleic acid sequence of SEQ ID NO:
14, or a nucleic acid
sequence at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence identity to
SEQ ID NO: 14.
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[00437] In some embodiments, the rAAV genotype comprises an intron sequence
selected in the
group consisting of a human beta globin b2 (or HBB2) intron, a FIX intron, a
chicken beta-globin
intron, and a SV40 intron. In some embodiments, the intron is optionally a
modified intron such as a
modified HBB2 intron (see, e.g., SEQ ID NO: 17 in of W02018046774A1): a
modified FIX intron
(see., e.g., SEQ ID NO: 19 in W02013046774A1), or a modified chicken beta-
globin intron (e.g., see
SEQ ID NO: 21 in W02018046774A1), or modified HBB2 or FIX introns disclosed in
W02015/162302, which are incorporated herein in their entirety by reference.
H. Poly-A
1004381 In some embodiments, an rAAV vector genome includes at least one poly-
A tail that is
located 3' and downstream from the heterologous nucleic acid gene encoding the
in one embodiment,
a GAA fusion polypeptide (es., SS-GAA fusion polypeptide or SS-IGF2-GAA
polypeptide). In some
embodiments, the polyA signal is 3' of a stability sequence or CS sequence as
defined herein. Any
polyA sequence can be used, including but not limited to hGH poly A, synpA
polyA and the like. In
some embodiments, the polyA is a synthetic polyA sequence. In some
embodiments, the rAAV vector
genome comprises two poly-A tails, e.g., a hGH poly A sequence and another
polyA sequence, where
a spacer nucleic acid sequence is located between the two poly A sequences. In
some embodiments,
the rAAV genome comprises 3' of the nucleic acid encoding the GAA fusion
polypeptide (e.g., SS-
GAA fusion polypeptide or SS-IGF2-GAA polypeptide), or alternatively, 3' of
the CS sequence the
following elements; a first polyA sequence, a spacer nucleic acid sequence (of
between 100-400bp, or
about 250bp), a second poly A sequence, a spacer nucleic acid sequence, and
the 3' ITR. In some
embodiments, the first and second poly A sequence is a hGH poly A sequence,
and in some
embodiments, the first and second poly A sequences are a synthetic poly A
sequence. In some
embodiments, the first poly A sequence is a hGH poly A sequence and the second
poly A sequence is
a synthetic sequence, or vice versa - that is, in alternative embodiments, the
first poly A sequence is a
synthetic poly A sequence and the second poly A sequence is a hGH polyA
sequence. An exemplary
poly A sequence is, for example, SEQ ID NO: 15 (hGH poly A sequence), or a
poly A nucleic acid
sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide
sequence identity
to SEQ ID NO: 15. In some embodiments, the hGHpoly sequence encompassed for
use is described in
Anderson et al. J. Biol. Chem 264(14); 8222-8229, 1989 (See, e.g. p. 8223, 2nd
column, first
paragraph) which is incorporated herein in its entirety by reference.
[00439] In some embodiments, a poly-A tail can be engineered to stabilize the
RNA transcript that
is transcribed from an rAAV vector genome, including a transcript for a
heterologous gene, which in
one embodiment is a GAA, and in alternative embodiments, the poly-A tail can
be engineered to
include elements that are destabilizing.
[00440] In some embodiments of the methods and compositions disclosed herein,
a recombinant
AAV vector comprises at least one polyA sequence located 3' of the nucleic
acid encoding the GAA
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gene and 5' of the 3' ITR sequence. In some embodiments, the poly A is a full
length poly A (II-
polyA) sequence. In some embodiments, the polyA is a truncated polyA sequence,
see. e.g., FIG. 5G.
[00441] In an embodiment, a poly-A tail can be engineered to become a
destabilizing element by
altering the length of the poly-A tail. In an embodiment, the poly-A tail can
be lengthened or
shortened. In some embodiments, the 3' untranslated region comprises GAA 3'
UTR (SEQ ID NO:
85) or a 3' UTR (SEQ ID NO: 77).
[00442] In another embodiment, a destabilizing element is a microRNA (miRNA)
that has the
ability to silence (repress translation and promote degradation) the RNA
transcripts the miRNA binds
to that encode a heterologous gene. In an embodiment, addition or deletion of
seed regions within
the poly-A tail can increase or decrease expression of a protein, such as the
GAA protein or modified
GAA polypeptide.
[00443] In another embodiment, seed regions can also be engineered into the 3'
untranslated regions
located between the heterologous gene and the poly-A tail. In a further
embodiment, the destabilizing
agent can be an siRNA. The coding region of the siRNA can be included in an
rAAV vector genome
and is generally located downstream, 3' of the poly-A tail.
[00444] In all aspects of the methods and compositions as disclosed herein,
the rAAV genome may
also comprise a Stuffer DNA nucleic sequence. An exemplary stuffer DNA
sequence is SEQ ID NO:
71, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99%
nucleotide sequence identity thereto. As shown in FIGS 7-8 and FIGS 9A-9E, the
stuffer sequence is
located 3 of the poly A tail, for example, and is located 5' of the '3 ITR
sequence. In some
embodiments, the stuffer DNA sequence comprises a synthetic polyadenylation
signal in the reverse
orientation.
[00445] In some embodiments, a sniffer nucleic acid sequence (also referred to
as a "spacer" nucleic
acid fragment, see FIGS 7-8) can be located between the poly A sequence and
the 3' ITR (i.e., a
stuffer nucleic acid sequence is located 3' of the polyA sequence and 5' of
the 3' ITR) (see, e.g., FIG.
7-8). Such a stuffer nucleic acid sequence can be about 30bp, 50pb, 75bp,
100bp, 150bp, 200bp,
250bp, 300bp or longer than 300bp. In some embodiments of the methods and
compositions as
disclosed herein, a sniffer nucleic acid fragment is between 20-50bp, 50-
100bp, 100-200bp, 200-
300bp, 300-500bp, or any integer between 20-500bp. Exemplary stuffer (or
spacer) nucleic acid
sequence comprise SEQ ID NO: 16, SEQ ID NO: 71 or SEQ ID NO: 78, or a nucleic
acid sequence at
least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99%, identical to SEQ ID NO: 16 or SEQ ID NO: 71 or SEQ ID
NO: 78.
I. AAV ITRs
1004461 The rAAV genome as disclosed here comprises AAV FIRs that have
desirable
characteristics and can be designed to modulate the activities of, and
cellular responses to vectors that
incorporate the ITRs. In another embodiment, the AAV ITRs are synthetic AAV
ITRs that has
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desirable characteristics and can be designed to manipulate the activities of
and cellular responses to
vectors comprising one or two synthetic Hilts, including, as set forth in U.S.
Patent No. 9,447433,
which is incorporated herein by reference.
1004471 In another embodiment, an ITR exhibits modified transcription activity
relative to a
naturally occurring ITR, e.g., ITR2 from AAV2. It is known that the ITR2
sequence inherently has
promoter activity. It also inherently has termination activity, similar to a
poly(A) sequence. The
minimal functional ITR of the present invention exhibits transcription
activity as shown in the
examples, although at a diminished level relative to ITR2. Thus, in some
embodiments, the ITR is
functional for transcription. In other embodiments, the ITR is defective for
transcription. In certain
embodiments, the ITR can act as a transcription insulator, e.g., preventing
transcription of a transgenic
cassette present in the vector when the vector is integrated into a host
chromosome.
1004481 One aspect of the invention relates to an rAAV vector genome
comprising at least one
synthetic AAV ITR, wherein the nucleotide sequence of one or more
transcription factor binding sites
in the ITR is deleted and/or substituted, relative to the sequence of a
naturally occurring AAV ITR
such as ITR2. In some embodiments, it is the minimal functional VTR in which
one or more
transcription factor binding sites are deleted and/or substituted. In some
embodiments at least 1
transcription factor binding site is deleted and/or substituted, e.g., at
least 5 or more or 10 or more
transcription factor binding sites, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or 21 transcription factor binding sites.
1004491 Another embodiment, a rAAV vector, including an rAAV vector genome as
described
herein comprises a polynucleotide comprising at least one synthetic AAV ITR,
wherein one or more
CpG islands (a cytosine base followed immediately by a guanine base (a CpG) in
which the cytosines
in such arrangement tend to be methylated) that typically occur at, or near
the transcription start site in
an ITR are deleted and/or substituted. In an embodiment, deletion or reduction
in the number of CpG
islands can reduce the inununogenicity of the rAAV vector. This results from a
reduction or complete
inhibition in TLR-9 binding to the rAAV vector DNA sequence, which occurs at
CpG islands. It is
also well known that methylation of CpG motifs results in transcriptional
silencing. Removal of CpG
motifs in the ITR is expected to result in decreased TLR-9 recognition and/or
decreased methylation
and therefore decreased transgene silencing. In some embodiments, it is the
minimal functional ITR
in which one or more CpG islands are deleted and/or substituted. In an
embodiment, AAV ITR2 is
known to contain 16 CpG islands of which one or more, or all 16 can be
deleted.
1004501 In some embodiments, at least 1 CpG motif is deleted and/or
substituted, e.g., at least 4 or
more or 8 or more CpG motifs, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 16 CpG
motifs.
1004511 In another embodiment, the synthetic ITR comprises, consists
essentially of, or consists of
one of the nucleotide sequences listed in Table 7. In other embodiments, the
synthetic ITR comprises,
consist essentially of, or consist of a nucleotide sequence that is at least
80% identical, e.g., at least
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85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleotide
sequences listed in
Table 7. In some embodiments, the ITR is a sequence is disclosed in FIG. 1 of
Samulski et at, 1983,
Cell, 33; 135-143 (referred to "Samulski eta!, 1983" as which is incorporated
herein in its entirety by
reference), which discloses modified ITR sequences in FIG. 1. In some
embodiments, the ITR
sequence comprises, or consists of a nucleotide sequence that is at least 80%
identical, e.g., at least
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of the ITR. sequences in
FIG. 1 as disclosed
in Samulski et al, 1993. In some embodiments, the ITR comprises, or consists
of a nucleotide
sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%,
97%, 98%, or 99% or
99.5% identical to the ITR sequence of pSM 609 right disclosed in the middle
panel of FIG. 1 (that
lacks the 9bp) disclosed in Samulski et al, 1983. In some embodiments, the ITR
comprises a
nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%,
95%, 96%, 97%, 98%, or
99% or 99.5% identical to the ITR sequence of any of SEQ ID NOs: 441-444. In
some embodiments,
the ITR sequence, e.g., Right ITR (or 3' ITR) is SEQ ID NO: 442 or a
nucleotide sequence that is at
least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or
99.5% identical to SEQ
ID NO: 442. In some embodiments, the ITR sequence, e.g., left ITR (or 5' ITR)
is SEQ ID NO: 441
or a nucleotide sequence that is at least 80% identical, e.g., at least 85%,
90%, 95%, 96%, 97%, 98%,
or 99% or 99.5% identical to SEQ ID NO: 441.
1004521 Table 7: Exemplary synthetic ITR sequences
MI-1-257
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
CTCACTGAGGCAATTTGATAAAAATCGTCAAATTATAAACAGGCTTTGCC
TOE II
CCATCACTAGGGGTTCCT (SEQ ID NO: 36)
MH-258
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
CTCACTGAGGGATAAAAATCCAGGCTTTGCCTGCCTCAGTGAGCGAGCG
AGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ
ID NO: 37)
MU Delta
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
258
CTCACTGAGGGATAAAAATCCAGGCTTTGCCTGCCTCAGTGAGCGAGCG
AGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG-GGTTCCT (SEQ
ID NO: 38)
MU Telomere- AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTOGGATTGGGATTG
1 ITR
CGCGCTCGCTCGCGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTG
ATAAAAATCAATCCCAATCCCAATCCCAATCCCAATCCCAATCCCCCGAG
CGAGCGCGCAATCCCAATCCCAGAGAGGGAGTGGCCAACTCCATCACTA
GGGGTTCCT (SEQ ID NO: 39)
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MU Telomere- AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
2 ITR
CTCGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGATAAAAATC
AATCCCAATCCCAATCCCAATCCCAATCCCAATCCCGCGAGCGAGCGCGC
AGGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTAAGCTTATTAT
A (SEQ ID NO: 40)
MU Poll! 258 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
ITR
CTCACTGAGGGCGCCTATAAAGATAAAAATCCAGGCTTTGCCTGCCTCAG
TTAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGG
TTCCT (SEQ ID NO: 41)
MR 258
CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGA
Delta D
GGGATAAAAATCCAGGCTTTGCCTGCCTCAGTGAGCGAGCGAGCGCGCA
conservative GAGAGGGAGTGGCCAACTCCATCACTAG (SEQ ID
NO: 42)
5' ITR
TTGGCCACTCCCTCTCTUCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC
AAAGGTCGCCCGACGCCCGGGCTITGCCCGGGCGGCCTCAGTGAGCGAG
CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ
ID NO: 161)
3' ITR AGGAAC
CCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCC
GGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
(SEQ ID NO: 165)
L-ITR
CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTC
GGGCGACCITTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA
GGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 441)
R-ITR
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTITGCCC
GGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 442)
ITR (145bp)
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG
CTCACTGAGGCCOGGCGACCAAAGGTCGCCCGACGCCCGGGC111GCCC
GGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
(SEQ ID NO: 443)
ITR (145bp ¨ AGGAAC CCCTAGTGATGGAGTTGGCCACTCCCTCTC
TGCGCGCTCGC TCG
1983 l acking
CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCC
,
GGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGG (SEQ ID NO: 444)
9bp)
J. Exemplary rAAV genome elements:
(i) AAV-LVR412
1004531 In some embodiments, the rAAV comprises in its genome, a construct
comprising in a 5' to
3' direction, a5' AAV2-ITR (SEQ ID NO: 161), stuffer DNA (SEQ ID NO: 162),
5130412 LSP (SEQ
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ID NO: 91), Kozak sequence for Glue (SEQ ID NO: 153), a nucleic acid sequence
encoding GAA
polypeptide (SEQ ID NO: 182), a 3' UTR (SEQ ID NO: 77), poly A sequence (SEQ
ID NO: 164), a
3' AAV-ITR sequence (SEQ ID NO: 165). An exemplary construct is shown as SEQ
ID NO: 154
(LVR412 AskBioEU). One can readily change the ITRs sequences of SEQ ID NO: 161
and 165 for
any ITR sequences for different AAV serotypes, or for ITRs selected from any
of SEQ ID Nos: 441-
444, as well as use a different UTR sequence or polyA sequence instead of SEQ
ID NO: 77 and SEQ
ID NO: 164, respectively.
1004541 In some embodiments, the rAAV comprises in its genome, a construct
comprising in a 5' to
3' direction, a 5' AAV2-ITR (SEQ ID NO: 161), stuffer DNA (SEQ ID NO: 162),
5P0412 LSP (SEQ
ID NO: 91), a nucleic acid sequence encoding GAA polypeptide (SEQ ID NO: 182),
a 3' UTR (SEQ
ID NO: 77), a poly A sequence (SEQ ID NO: 166), a 3' AAV-ITR sequence (SEQ ID
NO: 165). An
exemplary construct is shown as SEQ ID NO: 155 (ssAAV_LVR412WT-
hGAA_AskBio_CHATHAM_Backbone_Ask). One can readily change the ITRs sequences
of SEQ
ID NO: 161 and 165 for any ITR sequences for different AAV serotypes, or for
ITRs selected from
any of SEQ ID Nos: 441-444, as well as use a different UTR sequence or polyA
sequence instead of
SEQ ID NO: 77 and SEQ ID NO: 164, respectively.
1004551 AAV2-LVP422:
1004561 In some embodiments, the rAAV comprises in its genome, a construct
comprising in a 5' to
3' direction, a 5' AAV2-ITR (SEQ ID NO: 161), sniffer DNA, SP0422 LSP (SEQ ID
NO: 92), Kozak
sequence for Glue (SEQ ID NO: 153), a nucleic acid sequence encoding GAA
polypeptide (SEQ ID
NO: 55), a collagen stability sequence (SEQ ID NO: 65), a poly A sequence (SEQ
ID NO: 164), a 3'
AAV-ITR sequence (SEQ ID NO: 165). An exemplary construct is shown as SEQ ID
NO: 156
(LVR412Stuffer). One can readily change the ITRs sequences of SEQ ID NO: 161
and 165 for any
ITR sequences for different AAV serotypes, or for ITIts selected from any of
SEQ ID Nos: 441-444
as well as use a different collagen stability sequence or polyA sequence
instead of SEQ ID NO: 65
and SEQ ID NO: 164, respectively.
1004571 In some embodiments, the rAAV comprises in its genome, a construct
comprising in a 5' to
3' direction, a 5' AAV2-ITR (SEQ ID NO: 161), stuffer DNA, SP0422 LSP (SEQ ID
NO: 92), Kozak
sequence for Glue (SEQ ID NO: 153), a nucleic acid sequence encoding GAA
polypeptide (SEQ ID
NO: 182), a 3' UTR sequence (SEQ ID NO: 77), a poly A sequence (SEQ ID NO:
164), optionally
comprising a AATAA stop signal, a 3' AAV-ITR sequence (SEQ ID NO: 165). An
exemplary
construct is shown as SEQ ID NO: 157 (LVR422AskBio EU construct). One can
readily change the
ITRs sequences of SEQ ID NO: 161 and 165 for any ITR sequences for different
AAV serotypes or
for nRs selected from any of SEQ ID Nos: 441-444, as well as use a different
UTR sequence or
polyA sequence instead of SEQ ID NO: 77 and SEQ ID NO: 164, respectively
1004581 One can readily change the ITRs sequences in the rAAV genomes of SEQ
ID Nos: 160,
159, 155, 158, 156 for any ITR sequences for different AAV serotypes or for
ITRs of SEQ ID NOs:
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441-444, as well as use a different UTR sequence or polyA sequence, as
disclosed herein.
1004591In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence encoding a GAA polypeptide comprising SEQ ID NO: 170 (GAA
polypeptide with a
cognate GAA signal sequence and H199R_, R223H modifications), or SEQ ID NO:
171 (GAA
polypeptide with a cognate GAA signal sequence and H199R, H201L and R223H
modifications). The
GAA polypeptide of SEQ ID NO: 170 is encoded by the nucleic acid sequence of
SEQ ID NO: 182.
Accordingly, in some embodiments, the rAAV vector comprises a nucleic acid of
SEQ ID NO: 182
encoding a modified GAA polypeptide comprising H199R, R223H modifications. The
GAA
polypeptide of SEQ ID NO: 171 is encoded by the nucleic acid sequence of SEQ
ID NO: 182 where
basepairs (bp) 667-669 of SEQ ID NO: 182 are changed from CAC to any of: UUA,
UUG, CUU,
CUC CUA, CUG (resulting in a Histadine (H) to Leucine (L) amino acid change);
or where bp 668 of
SEQ ID NO: 182 is changed from A to U (resulting in a Histadine (H) to Leucine
(L) amino acid
change). Accordingly, in some embodiments, the rAAV vector comprises a nucleic
acid of SEQ ID
NO: 182, where bp 667-669 of SEQ ID NO: 182 are changed from CAC to any of:
UUA, UUG,
CUU, CUC CUA, CUG); or where bp 668 of SEQ ID NO: 182 is changed from A to U,
which
encodes a modified GAA polypeptide comprising H199R, H201L and R223H
modifications.
1004601In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence encoding a GAA polypeptide selected from any of: SEQ ID NO: 172
(GAA
polypeptide where cognate signal peptide is replaced with a IgG signal
sequence and H199R, R223H
modifications), or a sequence at least 85% sequence identity to SEQ ID NO:
172, or SEQ ID NO: 173
(GAA polypeptide where cognate signal peptide is replaced with a wtIL2 signal
sequence and H199R
and R223H modifications), or a sequence at least 85% sequence identity to SEQ
ID NO: 173, or SEQ
ID NO: 174 (GAA polypeptide where cognate signal peptide is replaced with a
mutIL3 signal
sequence and H199R and R223H modifications) or a sequence at least 85%
sequence identity to SEQ
ID NO: 174.
1004611In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 177 (IgG signal sequence), which encodes a GAA
polypeptide of SEQ
ID NO: 172 (IgG leader-GAA with H199R and R223H modifications). In some
embodiments, the
rAAV vector comprises a heterologous nucleic acid sequence comprising SEQ ID
NO: 182, where bp
668 of SEQ ID NO: 182 is changed from A to U and where bp 1-81 of SEQ ID NO:
182 is replaced
with the nucleic acid of SEQ ID NO: 177 (IgG signal peptide), or a sequence at
least 85% sequence
identity thereto, which encodes a GAA polypeptide of SEQ ID NO: 172 (IgG
leader-GAA with
H199R, H201L and R223H modifications).
1004621In some embodiments, the rAAV vector or rAAV genome comprises a
heterologous nucleic
acid sequence comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is
replaced with the
nucleic acid of SEQ ID NO: 179 (wt IL2 signal peptide), or a sequence at least
85% sequence identity
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thereto, which encodes a (MA polypeptide of SEQ ID NO: 173 (wt IL2 signal
peptide-GAA with
H199R, R223H modifications). In some embodiments, the rAAV vector comprises a
heterologous
nucleic acid sequence comprising SEQ ID NO: 182, where bp 668 of SEQ ID NO:
182 is changed
from A to U and where bp 1-81 of SEQ ID NO: 182 is replaced with the nucleic
acid of SEQ ID NO:
179 (wt IL2 signal peptide), or a sequence at least 85% sequence identity
thereto, which encodes a
GAA polypeptide of SEQ ID NO: 173 (wt IL2 signal peptide-GAA with H199R, H201L
R223H
modifications).
1004631In some embodiments, the rAAV vector comprises a heterologous nucleic
acid sequence
comprising SEQ ID NO: 182 where bp 1-81 of SEQ ID NO: 182 is replaced with the
nucleic acid of
SEQ ID NO: 181 (mutIL2 signal peptide), or a sequence at least 85% sequence
identity thereto, which
encodes a GAA polypeptide of SEQ ID NO: 174 (mutIL2 signal peptide-GAA with
11199R, R223H
modifications). In some embodiments, the rAAV vector comprises a heterologous
nucleic acid
sequence comprising SEQ ID NO: 182, where bp 668 of SEQ ID NO: 182 is changed
from A to U
and where bp 1-81 of SEQ ID NO: 182 is replaced with the nucleic acid of SEQ
ID NO: 181 (mut IL2
signal peptide), or a sequence at least 85% sequence identity thereto, which
encodes a (IAA
polypeptide of SEQ ID NO: 174 (min IL2 signal peptide-GAA with H199R, H201Land
R223H
modifications).
III. Vectors and Virions
1004641 In one embodiment, the rAAV vector (also referred to as a rAAV virion)
as disclosed herein
comprises a capsid protein, and a rAAV genome in the capsid protein. A rAAV
capsid of the rAAV
virion used to treat Pompe Disease is any of those listed in Table 1 disclosed
in 62,937,556, filed on
November 19, 2019, which is incorporated herein in its entirety by reference,
or any combination
thereof.
1004651 In one embodiment, a rAAV capsid of the rAAV virion used to treat
Pompe Disease is any
of those listed in Table 1 as disclosed in International Applications
W02020/102645, and
W02020/102667, each of which are incorporated herein in their entirety. In one
embodiment, a
rAAV capsid of the rAAV virion used to treat Pompe Disease is an AAV8 capsid.
In one
embodiment, a rAAV vector is an rAAV8 vector.
1004661 In one embodiment, the AAV vector (also referred to as a rAAV virion)
as disclosed herein
comprises a capsid protein from any of those disclosed in W02019/241324, which
is specifically
incorporated herein in its entirety by reference. In some embodiments, the
rAAV vector comprises a
liver specific capsid, e.g., a liver specific capsid selected from XL32 and
XL32.1, as disclosed in
W02019/241324, which is incorporated herein in its entirety by reference. In
some embodiments, the
rAAV vector is a AAVXL32 or AAVXL32.1 as disclosed in W02019/241324, which is
incorporated
herein in its entirety by reference.
1004671 Exemplary chimeric or variant capsid proteins that can be used as the
AAV capsid in the
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rAAV vector described herein can be selected from Table 2 from U.S.
provisional application
62,937,556, filed on November 19, 2019, which is specifically incorporated
herein in its reference, or
can be used with any combination with wild type capsid proteins and/or other
chimeric or variant
capsid proteins now known or later identified and each is incorporated herein.
In some embodiments,
the rAAV vector encompassed for use is a chimeric vector, e.g., as disclosed
in 9,012,224 and US
7,892,809, which are incorporated herein in their entirety by reference.
1004681 In some embodiments, the rAAV vector is a haploid rAAV vector, as
disclosed in US
application US2018/0371496 and PCT/U518/22725, or polyploid rAAV vector, e.g.,
as disclosed in
PCT/U52018/044632 filed on 7/31/2018 and in US application 16/151,110, each of
which are
incorporated herein in their entirety by reference. In some embodiments, the
rAAV vector is a
rAAV3 vector, as disclosed in 9,012,224 and WO 2017/106236 which are
incorporated herein in their
entirety by reference.
1004691 In a particular embodiment, the rAAV is a AAVXL32 or AAVXL32.1 AAV
vector as
disclosed in W02019/241324, which is incorporated herein in its entirety by
reference. In some
embodiments, the rAAV vector comprises a capsid disclosed in W02019241324A1,
or International
Patent application PCT/U52019/036676, which are incorporated herein in their
entirety by reference.
In some embodiments, the AAV vector is a AAV8 vector or a rational haploid
comprising an AAV8
capsid protein. In some embodiments, the recombinant AAV vector is a chimeric
AAV vector,
haploid AAV vector, a hybrid AAV vector or polyploid AAV vector. In some
embodiments, the
recombinant AAV vector is a rational haploid vector, a mosaic AAV vector, a
chemically modified
AAV vector, or a AAV vector from any AAV serotypes, for example, from any AAV
serotype
disclosed in Table 1 as disclosed in International Applications W02020/102645,
and
W02020/102667, each of which are incorporated herein in their entirety.
1004701ln some embodiments, the AAV vector comprises a capsid which is encoded
by a nucleic acid
AAV capsid coding sequence that is at least 90% identical to a nucleotide
sequence of any one of
SEQ ID NOs: 1-3 as disclosed in W02019241324A1; or (b) a nucleotide sequence
encoding any one
of SEQ ID NOS:4-6 as disclosed in W02019241324A1. In some embodiments, an AAV
capsid
comprises an amino acid sequence at least 90% identical to any one of SEQ ID
NOS:4-6 as disclosed
in W02019241324A1, along with AAV particles comprising an AAV vector genome
and the AAV
capsid of the invention.
1004711 In one embodiment, the rAAV vector as disclosed herein comprises a
capsid protein,
associated with any of the following biological sequence files listed in the
file wrappers of USPTO
issued patents and published applications, which describe chimeric or variant
capsid proteins that can
be incorporated into the AAV capsid of this invention in any combination with
wild type capsid
proteins and/or other chimeric or variant capsid proteins now known or later
identified (for
demonstrative purposes, 11486254 corresponds to U.S. Patent Application No.
11/486,254 and the
other biological sequence files are to be read in a similar manner): 11486254,
11932017, 12172121,
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12302206, 12308959, 12679144, 13036343, 13121532, 13172915, 13583920,
13668120, 13673351,
13679684, 14006954, 14149953, 14192101, 14194538, 14225821, 14468108,
14516544, 14603469,
14680836, 14695644, 14878703, 14956934, 15191357, 15284164, 15368570,
15371188, 15493744,
15503120, 15660906, and 15675677.
[00472] In an embodiment, the AAV capsid proteins and virus capsids of this
invention can be
chimeric in that they can comprise all or a portion of a capsid subunit from
another virus, optionally
another parvovirus or AAV, e.g., as described in international patent
publication WO 00/28004,
which is incorporated by reference. In some embodiments, an rAAV vector genome
is single stranded
or a monomeric duplex as described in U.S. Patent No. 8,784,799, which is
incorporated herein.
[00473] As a thither embodiment, the AAV capsid proteins and virus capsids of
this invention can
be polyploid (also referred to as haploid) in that they can comprise different
combinations of VP!,
VP2 and VP3 AAV serotypes in a single AAV capsid as described in US
application
US2018/0371496, which is incorporated by reference.
[00474] In an embodiment, an rAAV vector useful in the treatment of Pompe
Disease as disclosed
herein is an AAV3b capsid. AAV3b capsids encompassed for use are described in
2017/106236, and
9,012,224 and 7,892,809, and International application PCT/US19/61653, filed
Nov 15, 2019, and
International Applications W02020/102645, and W02020/102667, each of which are
incorporated
herein in their entirety.
[00475] In some embodiments, the AAV3b capsid comprises SEQ ID NO: 44. In an
embodiment,
the AAV capsid used in the treatment of Pompe Disease can be a modified AAV
capsid that is
derived in whole or in part from the AAV capsid set forth in SEQ ID NO: 44. In
some embodiments,
the amino acids from an AAV3b capsid as set forth in SEQ ID NO: 44 can be, or
are substituted with
amino acids from another capsid of a different AAV serotype, wherein the
substituted and/or inserted
amino acids can be from any AAV serotype, and can include either naturally
occurring or partially or
completely synthetic amino acids.
[00476] In another embodiment, an AAV capsid used in the treatment of Pompe
Disease is an
AAV36265D capsid. In this particular embodiment, an AAV3b265D capsid comprises
a
modification in the amino acid sequence of the two-fold axis loop of an AAV3b
capsid via
replacement of amino acid 6265 of the AAV3b capsid with D265. In some
embodiments, an
AAV36265D capsid comprises SEQ ID NO: 46. However, the modified virus capsids
of the
invention are not limited to AAV capsids set forth in SEQ ID NO: 46. In some
embodiments, the
amino acids from AAV3b265D as set forth in SEQ ID NO. 46 can be, or are
substituted with amino
acids from a capsid from an AAV of a different serotype, wherein the
substituted and/or inserted
amino acids can be from any AAV serotype, and can include either naturally
occurring or partially or
completely synthetic amino acids.
[00477] In another embodiment an rAAV vector useful in the treatment of Pompe
Disease as
disclosed herein is an AAV3b265D549A capsid. In this particular embodiment, an
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AAV3b265D549A capsid comprises a modification in the amino acid sequence of
the two-fold axis
loop of an AAV3b capsid via replacement of amino acid G265 of the AAV3b capsid
with D265 and
replacement of amino acid T549 of the AAV3b capsid with A549. In some
embodiments, an
AAV3b265D549A capsid comprises SEQ ID NO: 50. However, the modified virus
capsids of the
invention are not limited to AAV capsids set forth in SEQ ID NO: 50. In some
embodiments, the
amino acids from AAV3b265D549A as set forth in SEQ ID NO: 50 can be, or are
substituted with
amino acids from a capsid from an AAV of a different serotype, wherein the
substituted and/or
inserted amino acids can be from any AAV serotype, and can include either
naturally occurring or
partially or completely synthetic amino acids. In some embodiments, the amino
acids from
AAV3bSASTG (i.e., a AAV3b capsid comprising Q263A/T265 mutations) can be, or
are substituted
with amino acids from a capsid from an AAV of a different serotype, wherein
the substituted and/or
inserted amino acids can be from any AAV serotype, and can include either
naturally occurring or
partially or completely synthetic amino acids.
1004781 In another embodiment, an rAAV vector useful in the treatment of Pompe
Disease as
disclosed herein is an AAV3b549A capsid. In this particular embodiment, an
AAV3b549A capsid
comprises a modification in the amino acid sequence of the two-fold axis loop
of an AAV3b capsid
via replacement of amino acid T549 of the AAV3b capsid with A549. In some
embodiments, an
AAV3b549A capsid comprises SEQ ID NO: 52. However, the modified virus capsids
of the
invention are not limited to AAV capsids set forth in SEQ ID NO: 52. In some
embodiments, the
amino acids from AAV3b549A as set forth in SEQ ID NO: 52 can be, or are
substituted with amino
acids from a capsid from an AAV of a different serotype, wherein the
substituted and/or inserted
amino acids can be from any AAV serotype, and can include either naturally
occurring or partially or
completely synthetic amino acids.
1004791 In another embodiment, an rAAV vector useful in the treatment of Pompe
Disease as
disclosed herein is an AAV3bQ263Y capsid. In this particular embodiment, an
AAV3bQ263Y capsid
comprises a modification in the amino acid sequence of the two-fold axis loop
of an AAV3b capsid
via replacement of amino acid Q263 of the AAV3b capsid with Y263. In some
embodiments, an
AAV3b549A capsid comprises SEQ ID NO: 54. However, the modified virus capsids
of the
invention am not limited to AAV capsids set forth in SEQ ID NO: 54. In some
embodiments, the
amino acids from AAV3bQ263Y as set forth in SEQ ID NO: 54 can be, or are
substituted with amino
acids from a capsid from an AAV of a different serotype, wherein the
substituted and/or inserted
amino acids can be from any AAV serotype, and can include either naturally
occurring or partially or
completely synthetic amino acids.
1004801 In another embodiment, an rAAV vector useful in the treatment of Pompe
Disease as
disclosed herein is AAV3bSASTG serotype or comprises a AAV3bSASTG capsid. In
this particular
embodiment, an AAV3bSASTG capsid comprises a modification in the amino acid
sequence to
comprise a SASTG mutation, in particular, the AAV3b capsid was modified to
resemble AAV2
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Q263A/T265 subvariant by introducing these modifications at similar positions
in the AAV3b capsid
(as disclosed in Messina EL, et al., Adeno-associated viral vectors based on
serotype 3b use
components of the fibroblast growth factor receptor signaling complex for
efficient transduction.
Hum. Gene Then 2012 Oct: 23(10):1031-4, Piacentino III, Valentino, et al. "X-
linked inhibitor of
apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-
enhanced arleno-associated
viral vector." Human gene therapy 23.6 (2012): 635-646.which are both
incorporated herein in their
entirety by reference). Accordingly, in some embodiments, an rAAV vector
useful in the treatment of
Pompe Disease as disclosed herein is AAV3bSASTG serotype or comprises a
AAV3bSASTG capsid
comprising a AAV3b Q263A/T265 capsid. In some embodiments, the amino acids
from
AAV3bSASTG can be, or are substituted with amino acids from a capsid from an
AAV of a different
serotype, wherein the substituted and/or inseited amino acids can be from any
AAV serotype, and can
include either naturally occurring or partially or completely synthetic amino
acids.
1004811 In order to facilitate their introduction into a cell, an rAAV vector
genome useful in the
invention are recombinant nucleic acid constructs that include (1) a
heterologous sequence to be
expressed (in one embodiment, a polynucleotide encoding a GAA polypeptide) and
(2) viral sequence
elements that facilitate integration and expression of the heterologous genes.
The viral sequence
elements may include those sequences of an AAV vector genome that are required
in cis for
replication and packaging (e.g., functional IThs) of the DNA into an AAV
capsid. In an embodiment,
the heterologous gene encodes GAA, which is useful for correcting a GAA-
deficiency in a patient
suffering from Pompe Disease. In an embodiment, such an rAAV vector genome may
also contain
marker or reporter genes. In an embodiment, an rAAV vector genome can have one
or more of the
AAV3b wild-type (WT) cis genes replaced or deleted in whole or in part, but
retain functional
flanking ITR sequences.
1004821 In one embodiment, an rAAV vector as disclosed herein useful in the
treatment of Pompe
Disease comprises a rAAV genome as disclosed herein, encapsulated by an AAV3b
capsid. In some
embodiments, an rAAV vector as disclosed herein useful in the treatment of
Pompe Disease
comprises a rAAV genome as disclosed herein, encapsulated by any AAV3b capsid
selected from:
AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46), AAV3b ST
(5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50);
AAV36549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54), or a
AAV3bSASTG
(i.e., Q263A/T265) capsid.
1004831 In some embodiments of the methods and compositions as disclosed
herein, the rAAV
vector as disclosed herein comprises the nucleic acid sequences of any of
AAV_LVR412_EU (SEQ
ID NO: 154), ssAAV_LVR412WT-hGAA AskBio_CHATHAM (SEQ ID NO: 155), AAV-
LVR412Stuffer (SEQ ID NO: 156), AAV_LVR422_EU (SEQ ID NO: 157), AAV-
LVR422_Stuffer
(SEQ ID NO: 158), ssAAV_LVR412 WT-hGAA CHATHAM (SEQ ID NO: 159),
ssAAV_LSP_WT-hGAA-CHATHAM (SEQ ID NO: 160), or a nucleic acid sequence having
at least
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80%, 85%, 90%, 95% or 98% identity thereto. In some embodiments of the methods
and
compositions as disclosed herein, the rAAV vector that comprises a nucleic
acid sequence of any of:
SEQ ID NO: 154-160 can have the wtGAA sequence replaced by a modified GAA
nucleic acid
sequence as disclosed herein.
1004841 In some embodiments of the methods and compositions as disclosed
herein, the rAAV
vector as disclosed herein comprises the nucleic acid sequences of any of: SEQ
ID NO: 57 (AAT-
V43M-wtGAA (deltal-69aa)); SEQ ID NO: 58 (ratFN1-IGF2V43M-wtGAA (deltal-
69aa)); SEQ ID
NO: 59 (hFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA
(delta
1-69)); SEQ ID NO: 61 (FN lrat- IGFA2-7-wtGAA (delta 1-69)); SEQ ID NO: 62
(hFN1- IGFA2-7-
wtGAA (delta 1-69)), or a nucleic acid sequence having at least 80%, 85%, 90%,
95% or 98% identity
thereto. In some embodiments of the methods and compositions as disclosed
herein, the rAAV vector
comprises a nucleic acid sequence of any of: AAT_hIGF2-V43M wtGAA dell-
69_Stuffer.V02
(SEQ ID NO: 79); FIBrat hIGF2-V43M_wtGAA_del 1-69_Stuffer.V02 (SEQ ID NO: 80);
FIBhum hIGF2-V43M_wtGAA dell-69_Stuffer.V02 (SEQ ID NO: 81); AAT GILT_wtGAA
del 1-
69
Stuffer.V02 (SEQ ID NO: 82);
FIBrat_GILT_wtGAA_dell-69_Stuffer.V02 (SEQ ID NO: 83);
FIBhtun GILT wtGAA del 1-69 Stuffer.V02 (SEQ ID NO: 84) or a nucleic acid
sequence having at
least 80%, 85%, 90%, 95% or 98% identity thereto.
1004851 In some embodiments of the methods and compositions as disclosed
herein, the rAAV
vector as disclosed herein comprises the nucleic acid sequences of any of:
AAV_LVR412_EU (SEQ
ID NO: 154), ssAAV LVR412WT-hGAA AskBio CHATHAM (SEQ ID NO: 155), AAV-
LVR412Stuffer (SEQ ID NO: 156), AAV_LVR422_EU (SEQ ID NO: 157), AAV-
LVR422_Stuffer
(SEQ ID NO: 158), ssAAV_LVR412 WT-hGAA CHATHAM (SEQ ID NO: 159),
ssAAV LSP WT-hGAA-CHATHAM (SEQ ID NO: 160), or a nucleic acid sequence having
at least
80%, 85%, 90%, 95% or 98% identity thereto. In some embodiments of the methods
and
compositions as disclosed herein, the rAAV vector that comprises a nucleic
acid sequence of any of:
SEQ ID NO: 154-160 can have the wtGAA sequence replaced by a modified GAA
nucleic acid
sequence as disclosed herein
IV. Optimized rAAV Vector Genome
1004861In some embodiments of the methods and compositions as disclosed
herein, an optimized
rAAV vector genome is created from any of the elements disclosed herein and in
any combination,
including nucleic acid sequences encoding a promoter, an ITR, a poly-A tail,
elements capable of
increasing or decreasing expression of a heterologous gene, and in one
embodiment, a nucleic acid
sequence that is codon optimized for expression of GAA protein in vivo (i.e.,
coGAA or codon
optimized GAA) and optionally, one or more element to reduce immunogenicity.
Such an optimized
rAAV vector genome can be used with any AAV capsid that has tropism for the
tissue and cells in
which the rAAV vector genome is to be transduced and expressed.
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1004871 In some embodiments, rAAV genome lacks the AAV P5 promoter, which is
normally
located upstream of the liver-specific promoter as disclosed herein. Normally,
the P5 promoter
controls expression of the AAV rep/cap proteins during AAV replication. In
some embodiments, this
P5 promoter fragment is present in the rAAV vector as disclosed herein which
contains predicted
transcription factor binding sites, e.g., cyclic AMP-responsive element-
binding protein 3 (CREB3),
which can be activated by endoplasmic reticulum (ER)/Golgi stress (Sampieri
2019), activating
transcription factor 2 (ATF2), which is also involved in stress response
(Watson 2017), Nuclear
Receptor Subfamily 1 Group I Member 2 (NR1I2) (also known as Pregnane X
receptor [13X11) is
known to be enriched in liver, and is activated by pregnane steroids, rifampin
and other molecules
including dexamethasone (NR1I2_HGNC) (Xing 2020). Accordingly, in some
embodiments, a
fragment of the AAV P5 promoter in the rAAV genome is removed without
affecting the intended
performance of the GAA cassette. In some embodiments, the rAAV vector also
comprises a RNA
polymerase II termination sequence located between the polyA signal and the 3'
ITR. An exemplary
terminal sequence is SEQ ID NO: 439 which introduces two termination codons
and one restriction
site (e.g., XhoI) replaces TAG, and is located immediately downstream of the
last coding amino acids
of hGAA, and immediately located upstream of the 3' UTR.
AAV3b capsid modifications
10048811n some embodiments of the methods and compositions as disclosed
herein, an AAV3b
capsid for use in a rAAV vector as disclosed herein, has an amino acid
identity in the range of, e.g.,
about 75% to about 100%, about 80% to about 100%, about 85% to about 100%,
about 90% to about
100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%,
about 85% to
about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about
97%, about 80%
to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to
about 97%, to any
of AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46), AAV3b ST
(5663V-I-T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50);
AAV36549A capsid (SEQ ID NO: 52); AAV3bQ2631( capsid (SEQ ID NO: 54) or a
AAV3bSASTG
capsid (i.e., a AAV3b capsid comprising Q263A/T265 mutations) disclosed in
Nienaber et at, Hum.
Gen Ther, 2012, 23(10); 1031-42 and Piaeentino III, Valentino, et al. "X-
linked inhibitor of apoptosis
protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced
adeno-associated viral
vector." Human gene therapy 23.6 (2012): 635-646, both of which are
incorporated herein in their
entirety by reference. In yet other aspects of this embodiment, an AAV derived
from AAV3b has an
amino acid identity in the range of, ag. , about 75% to about 100%, about 80%
to about 100%, about
85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 75%
to about 99%,
about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about
95% to about
99%, about 75% to about 97%, about 80% to about 97%, about 85% to about 97%,
about 90% to
about 97%, or about 95% to about 97%, to any of the amino acid sequence for
AAV3b capsid (SEQ
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ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46), AAV3b ST (5663V+T492V) capsid
(SEQ ID
NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50); AAV3b549A capsid (SEQ ID NO:
52);
AAV3bQ263Y capsid (SEQ ID NO: 54) a AAV3bSASTG capsid (i.e., a AAV3b capsid
comprising
Q263A/T265 mutations) as disclosed in Nienaber et al., Hum. Gen Ther, 2012,
23(10); 1031-42, and
Piacentino III, Valentino, et al. "X-linked inhibitor of apoptosis protein-
mediated attenuation of
apoptosis, using a novel cardiac-enhanced adeno-associated viral vector."
Human gene therapy 23.6
(2012): 635-646.but the capsid still is a functionally active AAV protein.
1004891 In some embodiments of the methods and compositions as disclosed
herein, the AAV
serotype (e.g. AAV3b) comprises an SASTG mutation as described in Messina EL,
et al., Adeno-
associated viral vectors based on serotype 3b use components of the fibroblast
growth factor receptor
signaling complex for efficient transduction. Hum. Gene Titer. 2012 Oct
23(10):1031-42, which is
incorporated herein in its entirety by reference.
10049011n some embodiments of the methods and compositions as disclosed
herein, an AAV3b
capsid for use in a rAAV vector as disclosed herein, has, e.g., at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 01 50 contiguous amino acid deletions, additions,
and/or substitutions relative
to any of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D
capsid (SEQ
ID NO: 46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A
capsid (SEQ
ID NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO:
54), or a
AAV3bSASTG capsid (i.e., a AAV3b capsid comprising Q263A1T265 mutations) (as
disclosed in
Nienaber et at., Hum. Gen Ther, 2012, 23(10); 1031-42); or at most 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, or 50 contiguous amino acid deletions, additions, and/or
substitutions relative to
any of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D
capsid (SEQ ID
NO: 46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid
(SEQ ID
NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54) a
AAV3bSASTG capsid (i.e., a AAV3b capsid comprising Q263A1T265 mutations) (as
disclosed in
Nienaber et at., Hum. Gen Ther, 2012, 23(10); 1031-42). In yet another
embodiment, an AAV3b
capsid for use in a rAAV vector as disclosed herein, has, e.g., at least 1, 2,
3,4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, or 50 contiguous amino acid deletions, additions,
and/or substitutions relative
to any of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D
capsid (SEQ
ID NO: 46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A
capsid (SEQ
ID NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO:
54), or a
AAV3bSASTG capsid (i.e., a AAV3b capsid comprising Q263A/1265 mutations) (as
disclosed in
Nienaber et at., Hum. Gen Titer, 2012, 23(10); 1031-42); or at most 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, or 50 contiguous amino acid deletions, additions, and/or
substitutions relative to
any of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D
capsid (SEQ ID
NO: 46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid
(SEQ ID
NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54),
or a
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AAV36SASTG capsid (i.e., a AAV3b capsid comprising Q263A/T265 mutations) (as
disclosed in
Nienaber etal., Hum. Gen Thor, 2012, 23(10); 103142), but is still a
functionally active AAV.
[00491] In some embodiments of the methods and compositions as disclosed
herein, an AAV3b
capsid for use in a rAAV vector as disclosed herein, has an amino acid
identity in the range of, e.g.,
about 75% to about 100%, about 80% to about 100%, about 85% to about 100%,
about 90% to about
100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%,
about 85% to
about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about
97%, about 80%
to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to
about 97%, to any
of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid
(SEQ ID NO:
46), AAV3b ST (S663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ
ID NO:
50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ2631( capsid (SEQ ID NO: 54), a
AAV3bSASTG capsid(i.e., a AAV3b capsid comprising Q263A/T265 mutations) (as
disclosed in
Nienaber et at., Hum. Gen Thor, 2012, 23(10); 1031-42). In yet a fiirther
embodiment, an AAV3b
capsid for use in a rAAV vector as disclosed herein has an amino acid identity
in the range of, e.g.,
about 75% to about 100%, about 80% to about 100%, about 85% to about 100%,
about 90% to about
100%, about 95% to about 100%, about 75% to about 99%, about 80% to about 99%,
about 85% to
about 99%, about 90% to about 99%, about 95% to about 99%, about 75% to about
97%, about 80%
to about 97%, about 85% to about 97%, about 90% to about 97%, or about 95% to
about 97%, to any
of the amino acid sequence for AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid
(SEQ ID NO:
46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ
ID NO:
50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ2631( capsid (SEQ ID NO: 54), a
AAV3bSASTG capsid (i.e., a AAV3b capsid comprising Q263A/1'265 mutations) (as
disclosed in
Nienaber et al., Hum. Gen Ther, 2012, 23(10); 103142), but is still a
functionally active AAV.
V. Methods of Treatment
A. Pompe Disease
[00492] The recombinant AAV expressing GAA protein as disclosed herein can be
used in methods to
treat Pompe disease and other glycogen storage diseases (GSD). Pompe disease
is a tare genetic
disorder caused by a deficiency in the enzyme acid alpha-glucosidase (GAA),
which is needed to
break down glycogen, a stored form of sugar used for energy. Pompe disease is
also known as
glycogen storage disease type II, GSD II, type II glycogen storage disease,
glycogenosis type II, acid
maltase deficiency, alpha-1,4-glucosidase deficiency, cardiomegalia glycogenic
diffiisa, and cardiac
form of generalized glycogenosis. The build-up of glycogen causes progressive
muscle weakness
(myopathy) throughout the body and affects various body tissues, particularly
in the heart, skeletal
muscles, liver, respiratory and nervous system.
1004931The presenting clinical manifestations of Pompe disease can vary widely
depending on the
age of disease onset and residual GAA activity. Residual GALA activity
correlates with both the
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amount and tissue distribution of glycogen accumulation as well as the
severity of the disease.
Infantile-onset Pompe disease (less than 1% of normal GAA activity) is the
most severe form and is
characterized by hypotonia, generalized muscle weakness, and hypertrophic
cardionlyopathy, and
massive glycogen accumulation in cardiac and other muscle tissues. Death
usually occurs within one
year of birth due to cardiorespiratory failure. Juvenile-onset (1-10% of
normal GAA activity) and
adult-onset (10-40% of nonnal GAA activity) Pompe disease are more clinically
heterogeneous, with
greater variation in age of onset, clinical presentation, and disease
progression. Juvenile- and adult-
onset Pompe disease are generally characterized by lack of severe cardiac
involvement, later age of
onset, and slower disease progression, but eventual respiratory or limb muscle
involvement results in
significant morbidity and mortality. While life expectancy can vary, death
generally occurs due to
respiratory failure.
1004941 In any embodiment of the methods and compositions as disclosed herein,
a GAA enzyme
suitable for treating Pompe disease includes a wild-type human GAA, or a
fragment or sequence
variant thereof which retains the ability to cleave al-4 linkages in linear
oligosaccharides. In some
embodiments of the methods and compositions as disclosed herein, the GAA
protein encoded by a
wild type GAA nucleic acid sequence, e.g., SEQ ID NO: 11 or SEQ ID NO; 71 In
some
embodiments of the methods and compositions as disclosed herein, the GAA
protein is encoded by a
codon optimized GAA nucleic acid sequence, for example, for any one or more of
(1) enhanced
expression in vivo, (2) to reduce CpG islands or (3) reduce the innate immune
response. In some
embodiments of the methods and compositions as disclosed herein, the GAA
protein is encoded by a
codon optimized GAA nucleic sequence, for example, any nucleic acid sequence
selected from any
of: SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76 or SEQ ID
NO: 182, or a
nucleic acid sequence having at least 60%, or 70%, or 80%, 85% or 90% or 95%,
or 98%, or 99%
sequence identity to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:
76 or SEQ ID
NO: 182.
1004951In some embodiments of the methods and compositions as disclosed
herein, a rAAV vector as
described herein transduces the liver of a subject and secretes the hGAA
polypeptide into the blood,
which perfuses patient tissues where the hGAA polypeptide, with the assistance
of the fused IGF2-
sequence, is taken up by cells and transported to the lysosome, where the
enzyme acts to eliminate
material that has accumulated in the lysosomes due to the enzyme deficiency.
For lysosomal enzyme
replacement therapy to be effective, the therapeutic enzyme must be delivered
to lysosomes in the
appropriate cells in tissues where the storage defect is manifest.
B. Modulating GAA Levels In A Cell a vivo
1004961The nucleic acids, vector, and virions as described herein can be used
to modulate levels of
GAA in a cell. The method includes the step of administering to the cell a
composition including a
nucleic acid that includes a polynucleotide encoding GAA interposed between
two AAV ITRs. The
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cell can be from any animal into which a nucleic acid of the invention can be
administered.
Mammalian cells (e.g., humans, dogs, cats, pigs, sheep, mice, rats, rabbits,
cattle, goats, etc.) from a
subject with GAA deficiency are typical target cells for use in the invention.
In some embodiments,
the cell is a liver cell or a myocardial cell e.g., a myocardiocyte.
[004971ln an embodiment ex vivo delivery of cells transduced with rAAV vector
is disclosed herein.
In a further embodiment, ex vivo gene delivery may be used to transplant cells
transduced with a
rAAV vector as disclosed herein back into the host. In a thither embodiment,
ex vivo stem cell (e.g.,
mesenchymal stem cell) therapy may be used to transplant cells transduced with
a rAAV vector as
disclosed herein cells back into the host. In another embodiment, a suitable
ex vivo protocol may
include several steps.
[00498] In some embodiments, a segment of target tissue (e.g., muscle, liver
tissue) may be harvested
from the subject, and the rAAV vector described herein used to transduce a GAA-
encoding nucleic
acid into a host's cells. These genetically modified cells may then be
transplanted back into the host.
Several approaches may be used for the reintroduction of cells into the host,
including intravenous
injection, intraperitoneal injection, subcutaneous injection, or in situ
injection into target tissue.
Microencapsulation of modified ex vivo cells transduced or infected with an
rAAV vector as
described herein is another technique that may be used within the invention.
Autologous and
allogeneic cell transplantation may be used according to the invention.
[004991ln yet another embodiment, disclosed herein is a method of treating a
deficiency of GAA in a
subject, comprising administering to the subject a cell expressing GAA as
disclosed herein, in a
pharmaceutically acceptable carrier and in a therapeutically effective amount.
In some embodiments,
the subject is a human.
C. Increasing GAA Activity In A Subject
[00500] The nucleic acids, vectors, and virions as described herein can be
used to modulate levels of
functional GAA polypeptide in a subject, e.g., a human subject, or subject
with Pompe disease or at
risk of having Pompe disease. The method includes administering to the subject
a composition
comprising the rAAV vector, comprising the rAAV genome as described herein,
comprising a
heterologous nucleic acid encoding GAA interposed between two AAV ITRs, where
the hGAA is
linked to a signal peptide as described herein, and optionally a IGF2
targeting peptide as disclosed
herein. The subject can be any animal, e.g., mammals (e.g., human beings,
dogs, cats, pigs, sheep,
mice, rats, rabbits, cattle, goats, etc.) are suitable subjects. The methods
and compositions of the
invention are particularly applicable to GAA-deficient human subjects.
[00501] Furthermore, the nucleic acids, vectors, and virions described herein
may be administered to
animals including human beings in any suitable formulation by any suitable
method. For example, in
any embodiment of the methods and compositions as disclosed herein, an rAAV
vector, or rAAV
genome as disclosed herein can be directly introduced into an animal,
including through
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administration by oral, rectal, transmucosal, intranasal, inhalation (e.g.,
via an aerosol), buccal (e.g.,
sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in
ovo), parenteral (e.g.,
intravenous, subcutaneous, intradermal, intramuscular [including
administration to skeletal,
diaphragm and/or cardiac muscle], intradermal, intrapleural, intracerebral,
and intraarticular), topical
(e.g., to both skin and mucosal surfaces, including airway surfaces, and
transdermal administration),
intralymphatic, and the like, as well as direct tissue or organ injection
(e.g., to liver, skeletal muscle,
cardiac muscle, diaphragm muscle or brain) or other parenteral route depending
on the desired route
of administration and the tissue that is being targeted.
10050211n some embodiments, as the an rAAV vector, or rAAV genome comprises a
nucleic acid
sequence encoding GAA under the control of, or operatively linked to a liver
specific promoter, the
methods and compositions as disclosed herein can be administered via
intravenous or intramuscular
injection, where the rAAV vector, or rAAV genome will travel to the liver and
express the GAA
protein.
10050311n any embodiment of the methods and compositions as disclosed herein,
administration to a
muscle can be by any suitable method including intravenous administration,
intra-arterial
administration, and/or intra-peritoneal administration. Exemplary modes of
administration include
oral, rectal, transmucosal, intranasal, inhalation (e.g., via an aerosol),
buccal (e.g., sublingual),
vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo),
parenteral (e.g., intravenous,
subcutaneous, intradermal, intramuscular [including administration to
skeletal, diaphragm and/or
cardiac muscle], intradermal, intrapleural, intracerebral, and
intraarticular), topical (e.g., to both skin
and mucosal surfaces, including airway surfaces, and transdermal
administration), intralymphatic, and
the like, as well as direct tissue or organ injection (e.g., to liver,
skeletal muscle, cardiac muscle,
diaphragm muscle or brain). The most suitable route in any given case will
depend on the nature and
severity of the condition being treated and/or prevented and on the nature of
the particular vector that
is being used.
10050411n any embodiment of the methods and compositions as disclosed herein,
administration to
skeletal muscle according to the present invention includes but is not limited
to administration to
skeletal muscle in the limbs (e.g., upper arm, lower arm, upper leg, and/or
lower leg), back, neck,
head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits. Suitable
skeletal muscles that
can be injected are disclosed in U.S. provisional application 62,937,556,
filed on November 19, 2019,
which is incorporated herein its entirety by reference.
10050511n any embodiment of the methods and compositions as disclosed herein,
the rAAV vectors
and/or rAAV genome as disclosed herein are administered to the skeletal
muscle, liver, diaphragm,
costal, and/or cardiac muscle cells of a subject. For example, a conventional
syringe and needle can
be used to inject a rAAV virion suspension into an animal. Parenteral
administration of a the rAAV
vectors and/or rAAV genome, by injection can be performed, for example, by
bolus injection or
continuous infusion. Formulations for injection may be presented in unit
dosage form, for example,
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in ampoules or in multi-close containers, with an added preservative. The
compositions may take such
forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain agents for a
pharmaceutical formulation, such as suspending, stabilizing and/or dispersing
agents. Alternatively,
the rAAV vectors and/or rAAV genome as disclosed herein can be in powder form
(e.g., lyophilized)
for constitution with a suitable vehicle, for example, sterile pyrogen-free
water, before use.
1005061In particular embodiments, more than one administration (e.g., two,
three, four, five, six,
seven, eight, nine, 10, etc., or more administrations) may be employed to
achieve the desired level of
gene expression over a period of various intervals, e.g, hourly, daily,
weekly, monthly, yearly, eta
Dosing can be single dosage or cumulative (serial dosing), and can be readily
determined by one
skilled in the art. For instance, treatment of a disease or disorder may
comprise a one-time
administration of an effective dose of a pharmaceutical composition virus
vector disclosed herein.
Alternatively, treatment of a disease or disorder may comprise multiple
administrations of an effective
dose of a virus vector carried out over a range of time periods, such as,
e.g., once daily, twice daily,
trice daily, once every few days, or once weekly.
1005071The timing of administration can vary from individual to individual,
depending upon such
factors as the severity of an individual's symptoms. For example, an effective
dose of a virus vector
disclosed herein can be administered to an individual once every six months
for an indefinite period
of time, or until the individual no longer requires therapy. A person of
ordinary skill in the art will
recognize that the condition of the individual can be monitored throughout the
course of treatment and
that the effective amount of a virus vector disclosed herein that is
administered can be adjusted
accordingly.
1005081 Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to injection,
or as emulsions.
Alternatively, one may administer the virus vector and/or virus capsids of the
invention in a local
rather than systemic manner, for example, in a depot or sustained-release
formulation. Further, the
virus vector and/or virus capsid can be delivered adhered to a surgically
implantable matrix (e.g., as
described in U.S. Patent Publication No. US-2004-0013645-A1). The virus
vectors and/or virus
capsids disclosed herein can be administered to the lungs of a subject by any
suitable means,
optionally by administering an aerosol suspension of respirable particles
comprised of the virus
vectors and/or virus capsids, which the subject inhales. The respirable
particles can be liquid or solid.
Aerosols of liquid particles comprising the virus vectors and/or virus capsids
may be produced by any
suitable means, such as with a pressure-driven aerosol nebulizer or an
ultrasonic nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729.
Aerosols of solid particles
comprising the virus vectors and/or capsids may likewise be produced with any
solid particulate
medicament aerosol generator, by techniques known in the pharmaceutical art.
1005091In some embodiments, the rAAV vectors and/or rAAV genome as disclosed
herein can be
formulated in a solvent, emulsion or other diluent in an amount sufficient to
dissolve an rAAV vector
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disclosed herein. In other aspects of this embodiment, the rAAV vectors and/or
rAAV genome as
disclosed herein can herein may be formulated in a solvent, emulsion or a
diluent in an amount of,
e.g., less than about 90% (v/v), less than about 80% (v/v), less than about
70% (v/v), less than about
65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than
about 50% (v/v), less than
about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less
than about 30% (v/v), less
than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v),
less than about 10% (v/v),
less than about 5% (v/v), or less than about 1% (v/v). In other aspects, the
rAAV vectors and/or
rAAV genome as disclosed herein can disclosed herein may comprise a solvent,
emulsion or other
diluent in an amount in a range of, e.g., about 1% (v/v) to 90% (v/v), about
1% (v/v) to 70% (v/v),
about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to
40% (v/v), about 1%
(v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v),
about 2% (v/v) to 50%
(v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2%
(v/v) to 20% (v/v), about
2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v), about 4% (v/v) to 40%
(v/v), about 4% (v/v) to
30% (v/v), about 4% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6%
(v/v) to 50% (v/v),
about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to
20% (v/v), about 6%
(v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to 40% (v/v),
about 8% (v/v) to 30%
(v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8%
(v/v) to 12% (v1v).
1005101In any embodiment of the methods and compositions as disclosed herein,
the rAAV vectors
and/or rAAV genome as disclosed herein, of any serotype, including but not
limited to encapsulated
by any AAV3b capsid selected from: AAV3b capsid (SEQ ID NO: 44); AAV3b265D
capsid (SEQ ID
NO: 46), AAV3b ST (5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid
(SEQ ID
NO: 50); AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54)
or
AAV3bSASTG capsid (i.e., a AAV3b capsid comprising Q263AfT265 mutations) can
comprise a
therapeutic compound in a therapeutically effective amount. In an embodiment,
as used herein,
without limitation, the term "effective amount" is synonymous with
"therapeutically effective
amount", "effective dose", or "therapeutically effective dose." In an
embodiment, the effectiveness of
a therapeutic compound disclosed herein to treat Pompe Disease can be
determined, without
limitation, by observing an improvement in an individual based upon one or
more clinical symptoms,
and/or physiological indicators associated with Pompe Disease. In an
embodiment, an improvement
in the symptoms associated with Pompe Disease can be indicated by a reduced
need for a concurrent
therapy.
11ifi5111To facilitate delivery of a rAAV vector and/or rAAV genome as
disclosed herein, it can be
mixed with a carrier or excipient. Carriers and excipients that might be used
include saline (especially
sterilized, pyrogen-free saline) saline buffers (for example, citrate buffer,
phosphate buffer, acetate
buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (for
example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,
and glycerol. USP
grade carriers and excipients are particularly useful for delivery of virions
to human subjects.
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1005121In addition to the formulations described previously, a rAAV vector
and/or rAAV genome as
disclosed herein can also be formulated as a depot preparation. Such long
acting formulations may be
administered by implantation (for example subcutaneously or intramuscularly)
or by IM injection.
Thus, for example, a rAAV vector and/or rAAV genome as disclosed herein may be
formulated with
suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives.
1005131In any embodiment of the methods and compositions as disclosed herein,
the method is
directed to treating Pompe Disease that results from a deficiency of GAA in a
subject, wherein a
rAAV vector and/or rAAV genome as disclosed herein is administered to a
patient suffering from
Pompe Disease, and following administration, GAA is secreted from cells in the
liver and there is
uptake of the secreted GAA by cells in skeletal muscle tissue, cardiac muscle
tissue, diaphragm
muscle tissue or a combination thereof, wherein uptake of the secreted GAA
results in a reduction in
lysosomal glycogen stores in the tissue(s). In some embodiments, the rAAV
vector and/or rAAV
genome as disclosed herein is encapsulated in a capsid, e.g., encapsulated by
any AAV3b capsid
selected from: AAV3b capsid (SEQ ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46),
AAV3b ST
(5663V+T492V) capsid (SEQ ID NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50);
AAV3b549A capsid (SEQ ID NO: 52); AAV3bQ263Y capsid (SEQ ID NO: 54).
1005141In a particular embodiment, at least about 102to about 108ce11s or at
least about 103to about
106 cells will be administered per dose in a pharmaceutically acceptable
carrier. In a further
embodiment, dosages of the virus vector and/or capsid to be administered to a
subject depend upon
the mode of administration, the disease or condition to be treated and/or
prevented, the individual
subject's condition, the particular virus vector or capsid, the nucleic acid
to be delivered, and the like,
and can be determined in a routine manner. Exemplary doses for achieving
therapeutic effects are
titers of at least about 105, 106, 107, 108, 109, 1010, 1011, 1012, 103, 1014,
1015 transducing units,
optionally about 108-10"transducing units.
1005151In another aspect, disclosed herein is a method of administering a
nucleic acid encoding a
GAA to a cell, comprising contacting the cell with a rAAV vector ancUor rAAV
genome as disclosed
herein, under conditions for the nucleic acid to be introduced into the cell
and expressed to produce
GAA. In some embodiments, the cell is a cultured cell. In some embodiments,
the cell is a cell in
vivo. In some embodiments, the cell is a mammalian cell. In some embodiments,
method of
administering a nucleic acid encoding a GAA to a cell further comprises
collecting the GAA secreted
into a cell culture medium.
D. Increasing Motoneuron Function In A Mammal
1005161In any embodiment of the methods and compositions as disclosed herein,
a rAAV vector
and/or rAAV genome as disclosed herein is useful in compositions and methods
to increase phrenic
nerve activity in a mammal having Pompe disease and/or insufficient GAA
levels. For example, a
rAAV vector and/or rAAV genome as disclosed herein, e.g., a rAAV vector and/or
rAAV genome
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encapsulated in a capsid, e.g., encapsulated by any AAV3b capsid selected
from: AAV3b capsid
(SEQ ID NO: 44); AAV3b265D capsid (SEQ ID NO: 46), AAV3b ST (S663V-FT492V)
capsid (SEQ
ID NO: 48), AAV3b265D549A capsid (SEQ ID NO: 50); AAV3b549A capsid (SEQ ID NO:
52);
AAV3bQ263Y capsid (SEQ ID NO: 54), can be administered to the central nervous
system (e.g.,
neurons). In another embodiment, retrograde transport of rAAV vector and/or
rAAV genome as
disclosed herein encoding GAA from the diaphragm (or other muscle) to the
phrenic nerve or other
motor neurons can result in biochemical and physiological correction of Pompe
disease. These same
principles could be applied to other neuroclegenerative disease.
1005171In an embodiment, a rAAV GAA construct of any scrotypc as described in
Table 1, including
AAV8 or AAV3, or AAV3b (including but not limited to AAV3b serotypes
AAV3b265D,
AAV3b265D549A, AAV3b549A, AAV3bQ263Y, AAV3bSASTG (i.e., a AAV3b capsid
comprising
Q263A/T265 mutations) serotypes) is capable of reducing any one or more of the
symptoms of (i) the
feeling of weakness in a patient's lower extremities, including, the legs,
trunk and/or arms, (ii) a
shortness of breath, a hard time exercising, lung infections, a big curve in
the spine, trouble breathing
while sleeping, an enlarged liver, an enlarged tongue and/or a stiff joint,
(iii) in a patient suffering
from Pompe Disease by, e.g., at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at
least 35%, at least 400/o, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as
compared to a patient
not receiving the same treatment. In other aspects of this embodiment, an AAV
GAA of any serotype
is capable of reducing any one or more of the systems of (i) the feeling of
weakness in a patient's
lower extremities, including, the legs, trunk and/or an-ris, ii) a shortness
of breath, a hard time
exercising, lung infections, a big curve in the spine, trouble breathing while
sleeping, an enlarged
liver, an enlarged tongue and/or a stiffjoint, (iii) in a patient suffering
from Pompe Disease by, e.g.,
about 10% to about 100%, about 20% to about 100%, about 30% to about 100%,
about 40% to about
100%, about 50% to about 100%, about 60% to about 100%, about 70% to about
100%, about 80% to
about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about
90%, about 40%
to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to
about 90%, about
10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to
about 80%,
about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%,
about 20% to about
70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%
as compared to
a patient not receiving the same treatment.
1005181In any embodiment of the methods and compositions as disclosed herein,
at least one
symptom associated with Pompe Disease, or at least one adverse side effect
associated with Pompe
Disease are reduced by at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at
least 75%, at least 80%, at least 35%, at least 90%, or at least 95%, and the
severity of at least one
symptom associated with Pompe Disease, or at least one adverse side effect is
reduced by at least
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10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least
85%, at least 90%, or at least 95%. In another embodiment, at least one
symptom associated with
Pompe Disease, or at least one adverse side effect associated with Pompe
Disease is reduced by about
10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40%
to about 100%,
about 50% to about 100%, about 60% to about 100%, about 70% to about 100%,
about 80% to about
100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%,
about 40% to
about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about
90%, about 10%
to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to
about 80%, about
50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20%
to about 70%,
about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.
E Immunosuppression
1005191 In any embodiment of the methods and compositions as disclosed herein,
a subject being
administered a rAAV vector or rAAV genome as disclosed herein is also
administered an
immunosuppressive agent. Various methods are known to result in the
immunosuppression of an
immune response of a patient being administered AAV. Methods known in the art
include
administering to the patient an immunosuppressive agent, such as a proteasome
inhibitor. One such
proteasome inhibitor known in the art, for instance as disclosed in U.S.
Patent No. 9,169,492 and U.S.
Patent Application No. 15/796,137, both of which are incorporated herein by
reference, is bortezomib.
In another embodiment, an immunosuppressive agent can be an antibody,
including polyclonal,
monoclonal, scfv or other antibody derived molecule that is capable of
suppressing the immune
response, for instance, through the elimination or suppression of antibody
producing cells. In a further
embodiment, the immunosuppressive element can be a short hairpin RNA (shRNA).
In such an
embodiment, the coding region of the shRNA is included in the rAAV cassette
and is generally located
downstream, 3' of the poly-A tail. The shRNA can be targeted to reduce or
eliminate expression of
immunostimulatory agents, such as cytokines, growth factors (including
transforming growth factors
131 and 132, TNF and others that are publicly known).
VI. Replacing GALA with other lysosomal enzymes.
10052.01 In some embodiments, in any of the methods and compositions as
disclosed herein, the
nucleic acid sequence encoding a (IAA polypeptide can be substituted for the
nucleic acid sequence of
a lysosomal enzyme. A lysosomal enzyme suitable to be expressed by the rAAV
vectors or rAAV
genomes as disclosed herein includes any enzyme that is capable of reducing
accumulated materials in
mammalian lysosomes or that can rescue or ameliorate one or more lysosomal
storage disease
symptoms. Suitable lysosomal enzymes include both wild-type or modified
lysosomal enzymes and
can be produced using recombinant or synthetic methods or purified from nature
sources. Exemplary
lysosomal enzymes are listed in Table 5A or Table 6A,
1005211 Table 5A: Exemplary Lysosomal Storage
Diseases (LSD) and associated enzyme
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defects
Disease I Enzyme defect
Substance stored
A. Glycogenosis Disorders
Pompe Disease Acid-a1,4-Glucosidase
Glycogen al-4 linked
Oli osaccharides
R Glycolipidosis Disorders
GM! Gangliodsidosis 13-Galactosidase
GMIGangliosides
Tay-Sachs Disease 13-Hexosaminidase A
GM2Ganglioside
AS Variant Protein
Sandhoff Disease P-Flexosaminidase A & B
GM2Ganglioside
Fabry Disease a-Galactosidase A
Globosides
Gaucher Disease Glucocerebrosidase
Glucosylcerami
Metachromatic Arylsulfata se A
Sulphatides
Leukodystrophy
Ksabbe Disease Galactosykeramidase
Galactocerebroside
Niematm-Pick, Types A & B Acid Sphingomyelinase
Sphingomyelin
Niemann-Pick, Type D Unknown
Sphingomyelin
Farber Disease Acid Ceramidase
Ceramide
Warn= Disease Acid Lipase
Cholesteryl Esters
C. Mucopolysaccharide disorders
Hurler Syndrome (MPS MI) a-L-Iduronidase
Heparan & Dennatan Sulfates
Scheie Syndrome (MPS IS) a-L-Iduronidase
Heparan & Dennatan Sulfates
Hurler-Scheie (MPS IH/S) a-L-Iduronidase
Heparan & Dermatan Sulfates
Hunter Syndrome (MPS II) Iduronate Sulfatase
Heparan & Dennatan Sulfates
Sanfilippo A (MPS IIIA) Heparan N-Sulfatase
Heparan Sulfate
Sanfilippo B (MPS MB) a-N-
Acetylglucosaminidase Heparan Sulfate
Sanfilippo C (MPS IIIC) Acetyl-CoA-
Glucosanainide Heparan Sulfate
Acetyltransferase
Sanfilippo D (MPS IIID) N-Acetylglucosamine-6-
Heparan Sulfate
Sulfatase
Morquio A (MPS WA) Galactosamine-6-
Sulfatase Keratan Sulfate
Morquio B (MPS IVB) ii-Galactosidase
Keratan Sulfate
Maroteaux-Lamy (MPS VI) Aiylsulfatase B
Dermatan Sulfate
Sly Syndrome (MPS VII) 13-Glucuronidase
D. Oligosaccharide/Glycoprotein Disorders
a-Mannosidosis a-Maimosidosis
Mannose/Oligosacharides
(3-Mannosidosis 13-Mannosidosis
Maimose/Oligosacharides
Fucosidosis a-L-Fucosidase
Fucosyl Oligosaccharides
Aspartylglucosaminuria N-Aspartyl-
Aspartylglucosamine
Glucosaminidase
Asparagines
Sialidosis (Mucolipidosis I) a-Neuraminidase
Sialyloligosacchatrides
Galactosialidosis (Goldberg Lysosomal Protective
Protein Sialyloligosaccharides
Syndrome) Deficiency
Schindler Disease a-N-Acetyl-
Galactosaminidase
E Lysosomal Enzyme Transport Disorders
Mucolipidosis II (I-Cell N-Acetylglucosamine-1-
Heparan Sulfate
Disease) Phosphotransferase
Mucolipidosis III (Pseudo- Same as MLII
Hurler Polydystrophy)
F. Lysosomal Membrane Transport Disorders
Cystinosis I Cystine Transport
Protein Free Cystine
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Saila Disease Sialic Acid Transport
Protein Free Sialic Acid and
Glucuronic Acid
Infantile Sialic Acid Sialic Acid Transport
Protein Free Sialic Acid and
Storage Disease
Glucuronic Acid
a Other
Batten Disease (Juvenile Unknown
Lipofuscins
Neuronal Ceroid
Lipofuscinosis)
Infantile Neuronal Palmitoyl-Protein
Lipofuscins
Ceroid Lipofuscinosis Thioesterase
Mucolipidosis IV Unknown
Gangliosides &Hyaluronic
Acid
Prosaposin Saposins A, B, C or D
1005221 In some embodiments of the composition and methods disclosed herein,
one particularly
preferred lysosomal enzyme is glucocerebrosidase, which is currently
recombinantly produced and
manufactured by Genzyme and used in enzyme replacement therapy for Gaucher's
Disease. Currently,
the recombinant enzyme is prepared with exposed mannose residues, which
targets the protein
specifically to cells of the macrophage lineage. Although the primary
pathology in type I Gaucher
patients are due to macrophage accumulating glucocerebroside, there can be
therapeutic advantage to
delivering glucocerebrosidase to other cell types. Targeting
glucocerebrosidase to lysosomes using the
present invention would target the agent to multiple cell types and can have a
therapeutic advantage
compared to other preparations. In some embodiments of the composition and
methods disclosed
herein, the lysosomal disease treated in the methods disclosed herein is not
Pompe. In some
embodiments of the composition and methods disclosed herein, the lysosomal
enzyme encoded by the
nucleic acid in the targeting vector or rAAV vector is not GAA.
1005231 While methods and compositions of the invention are useful for
producing and delivering
any therapeutic agent to a subcellular compartment, the invention is
particularly useful for delivering
gene products for treating metabolic diseases.
1005241 In some embodiments, a lysosomal enzyme for treating lysosomal storage
diseases (LSD)
are shown in Table 5A. In some embodiments, the lysosomal enzyme is associated
with Golgi or ER
defects, which are shown in Table 6A. In a preferred embodiment, a viral
vector encoding a
lysosomal enzyme as described herein is delivered to a patient suffering from
a defect in the same
lysosomal enzyme gene. In alternative embodiments, a functional sequence or
species variant of the
lysosomal enzyme gene is used. In further embodiments, a gene coding for a
different enzyme that
can rescue a lysosomal enzyme gene defect is according to methods of the
invention.
1005251 Table 6A: Diseases of the Golgi and ER
TABLE 6A: Diseases of the Golgi and ER
Disease Name Gene and
Features
Enzyme Defect
Ehlers-Danlos PLOD I lysyl
Defect in lysyl hydroxylation
Syndrome Type hydroxylase
of Collagen; located in ER
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VI
lumen
Type Ia glycogen glucose6 phosphatase
Causes excessive
storage disease
accumulation of Glycogen in
the liver, kidney, and
Intestinal mucosa; enzyme is
transmembrane but active site
is ER lumen
Congenital Disorders of Glycosvlation
CDG Ic ALG6
Defects in N-glycosylation ER
a1,3
lumen
glucosyltransf-erase
CDG Id ALG3
Defects in N-glycosylation ER
a1,3
transmembrane protein
mannosyltransferase
CDG IIa MGAT2
Defects in N-glycosylation
N-acetylglucosaminyl-
golgi transmembrane protein
transferase IT
CDG IR) GCS1
Defect in N glycosylation
a1,2-Glucosidase I
ER membrane bound with
lumenal catalytic domain
releasable by proteolysis
1005261 In some embodiments of the methods and compositions as disclosed
herein, a tnrgeting
vector or rAAV expresses a protein of any of the sequences in Table 5B or in
Table 6B.
1005271 Table 58: Exemplary Lysosomal Storage Diseases (LSD) and proteins to
be expressed by
targeting vectors or rAAV vectors, and nucleic acid sequences encoding the
proteins.
Disease Enzyme defect
Nucleic acid Protein sequence
sequence for
encoding the
expression by a
enzyme defect
targeting vector or
rAAV
A Glycogenosis Disorders
Pompe Disease Acid-a 1,4-Glucosidase
SEQ ID NO: 11 >> wt SEQ ID NO: 10 (aa
(GAA)(WT)
full length hGAA wt f-ull length hGAA
(non-codon
aa (non-codon
optimized);
optimized);NM_000
NP 000143.2
152.4), or SEQ ID
Nos 170, 171, 172,
173, 174
Acid-a 1,4-Glucosidase
SEQ ID NO: 72 or
(hGAA)
SEQ ID NO: 182>>
>hGAA
Acid-a1,4-Glucosidase
SEQ ID NO: 73
(hGAA 3X)
>hGAA 3X
hGAA
SEQ ID NO: 74
hGAA Codon_Optimize >hGAA_Codon_Opti
d No 1
mized No!
>hGAA Codon Optimiz SEQ ID NO: 75
ed No2
>hGAA_Coclon_Opti
mized_No2
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>hGAA_Codon_Optimiz SEQ ID NO: 76
ecl No3
>hGAA_Codon_Opti
mized_No3
B. Glyeolipidosis Disorders
GM1 Gangliodsidosis 13-Galactosidase (GLB1)
SEQ ID NO; 227 SEQ ID NO: 185
(GLB1 deficiency)
GLB1 NP 000395.3
(NM 000404.4)
Tay-Sachs Disease 13-Hexosaminidase A
SEQ ID NO: 228 SEQ ID NO: 186
(11E3CA)
NM_000520.6 NP_000511.2
(I1EXA)
(HEXA)
GM2-gangliosidosis, AB 0-11exosaminidase A
SEQ ID NO: 229 SEQ ID NO: 186
variant (HEXA) and HEXB
NM_000520.6 NP_000511.2
(HEXA)
(HEXA)
Sandhoff Disease 0-11exosaminidase A &
SEQ ID NO:229; SEQ ID NO: 186
I3-Hexosaminidase B
NM_000520.6 NP_000511.2
(HEXA and HEXB)
(HEXA) (HEXA)
SEQ ID NO: 230;
SEQ ID NO: 187;
NM_000521.4
NP_000512 .2(HEX
(I1EXB)
B)
Fabry Disease a-Galactosidase A (GLA) SEQ
ID NO: 231 SEQ ID NO: 188;
NM 000169.3 (GLA) NP 000160.1
(GLA)
Gaucher Disease Glucocerebrosidase
SEQ ID NO: 232 SEQ ID NO: 189;
(GBA)
NM 000157.4 (GBA) NP 000148.2
(GBA)
Metachromatic Arylsulfatase A (ARSA)
SEQ ID NO: 233 SEQ ID NO: 190
Leukodystrophy
NM_000487.6(ARSA)
Krabbe Disease (also Galactosylceramidase
SEQ ID NO: 234 SEQ ID NO: 191;
called globoid cell (GALC)
NP_000144.2
leukodystrophy)
Niemann-Pick, Types A & Acid Sphingomyelinase
SEQ ID NO: 235 SEQ ID NO: 192;
(SMPD1)
NM_000543.5 NP_000534.3
(SMPD1)
(SMPD1)
Niemann-Pick, Type Cl NPC intracellular
SEQ ID NO: 236 SEQ ID NO: 193;
cholesterol transporter 1
NM_000271.5 (NPC1) NP_000262.2
(NPC1)
(NPC1)
Niemann-Pick, Type C2 NPC intracellular
SEQ ID NO: 237 SEQ ID NO: 194;
cholesterol transporter 2
NM_006432.5 (NPC2) NP_006423.1
(NPC2)
(NPC2)
Farber Disease (Farber Acid Ceramidase
SEQ ID NO: 238 SEQ ID NO: 195;
lipogranulomatosis) (ASAH1)(also known as
NM_004315.6 NP_004306.3
N-acylsphingosine
(ASAH1) (ASAH1)
amidohydrolase)
Wolman Disease (also Lysomal Acid Lipase
SEQ ID NO: 239 SEQ ID NO: 196;
known as Lysosomal acid (LIPA) (also known as
NM_000235.4 (LIPA) NP_000226.2
lipase deficiency) Lipase A)
(LIPA)
C. Mucopolysaccharide disorders
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Mucopolysaccharidosis a-L-Iduronidase (IDUA)
SEQ ID NO: 240; SEQ ID NO: 197;
type I (MPS I) (includes 3
NM_000203.5 NP_000194.2
MPS I types: Hurler
(IDUA) (IDUA)
Syndrome (MPS IH);
Scheie Syndrome (MPS
IS) Hurler-Scheie (MPS
Mucopolysaccharidosis Iduronate Sulfatase (IDS) SEQ
ID NO: 241; SEQ ID NO: 198;
type II (MPS II), (also
NM_000202.8 (IDS) NP_000193.1 (IDS)
known as Hunter
syndrome)
Sanfilippo A (MPS IIIA) Heparan N-Sulfarase
SEQ ID NO: 242; SEQ ID NO: 199;
(also referred to as N-
NM_000199.5 NP_000190.1
sulfoglucosamine
(SGSH) (SGSH)
sulfohydrolase) (SGSH)
Sanfilippo B (MPS IIIB) a-N-
SEQ ID NO: 243; SEQ ID NO: 200
Acetylglucosaminidase
NM_000263,4 NP_000254.2
(NAGLU)
(NAGLU) (NAGLU)
Sanfilippo C (MPS IIIC) Acetyl-CoA-
SEQ ID NO: 244; SEQ ID NO: 201;
Glucosaminide
NM 152419.3 NP 689632 .2
Acetyltransferase (also
(HGSNAT) (HGSNAT)
referred to as heparan-
alpha-glucosaminide N-
acetyltransferase) (often
shortened to N-
acetyltransferase)
HGSNAT)
Sanfilippo D (MPS IIID) N-Acetylglucosamine-6-
SEQ ID NO: 245; SEQ ID NO: 202;
Sulfatase (GNS)(also
NM_002076.4 ((INS) NP_002067.1
referred to as
(GNS)
glucosamine (N-acetyl)-
6-sulfatase)
Mucopolysaccharidosis Galactosatnine-6-
SEQ ID NO: 246; SEQ ID NO: 203;
type WA (MPS WA) also Sulfatase (GALNS)
NM_000512 NP_000503.1
known as
(GALNS) (GALNS)
Morquio A syndrome)
Mucopolysaccharidosis 0-Galactosidase (GLB I)
SEQ ID NO: 227; SEQ ID NO: 185
type IVB (MPS IVB),
GLB1 NP_000395.3
(also known as Morquio B
(NM 000404.4)
syndrome)
Mucopolysaccharidosis Arylsulfatase B (ARSB)
SEQ ID NO: 247; SEQ ID NO: 204
type VI (MPS VI), (also
NM_0000465 NP_000037.2
known as Maroteaux-
(ARSB) (ARSB)
Lamy syndrome)
Mucopolysaccharidosis 13-Glucuronidase (GUSB) SEQ
ID NO: 248 SEQ ID NO: 205
type VII (MPS VII), (also
NM 000181.4 NP 000172.2
known as Sly syndrome)
(GUSB) (GUSB)
D. Oligosaccharide/Glycoprotein Disorders
a-Matmosidosis a-Maimosidosis
SEQ ID NO: 249 SEQ ID NO: 206
(MAN2B 1)
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NM_000528.4
NP_000519.2
(MAN2B1)
(MAN2B1)
13-Mannosidosis 13-Mannosidosis
SEQ ID NO: 250 SEQ ID NO: 207
(MANBA)
NM_005908.4 NP_005899.3
(MANBA)
(MANBA)
Fucosidosis a-L-Fucosidase (FUCA1) SEQ ID
NO: 251 SEQ ID NO: 208
NM_000147.4
NP_000138.2
(FUCA1)
(FUCA1)
Aspartylglucosaminuria N-Aspartyl- 13-
SEQ ID NO: 252 SEQ ID NO: 209
Glucosaminidase
NM_000027.4 (AGA) NP_000018.2
(AGA)(also known as
(AGA)
aspartylglucosaminidase
(ASRG)or N(4)-(beta-N-
acetylglucosaminy1)-L-
asparaginase or
glycosylasparaginase
(AGU)
Sialidosis (Mucolipidosis neuraminidase 1 (also
SEQ ID NO: 253
I) referred to as a-
NM_000434.4 SEQ ID NO: 210
Neuraminidase) (NEU1) (NEU1)
NP_000425.1
(NEU1)
Galactosialidosis (also Cathepsin A (CTSA)(also SEQ
ID NO: 254 SEQ ID NO: 211
known as Goldberg known as protective
NM_000308.4 NP_000299.3
Syndrome or Lysosomal protein/cathepsin A or
(CTSA) (CTSA)
Protective Protein PPCA or PPGB, or GSL)
Deficiency or PPCA
deficiency or
neuraminidase deficiency
with beta-galactosidase
deficiency)
Schindler Disease (also a-N-Acetyl-
SEQ ID NO: 255 SEQ ID NO: 212
referred to as NAGA Galactosaminidase
NM 000262.3 NP 000253 .1
deficiency or alpha- (NAGA)(also referred to
(NAGA) (NAGA)
galactosidase B a alpha-galactosidase B
deficiency) or GALB)
Lysosomal Enzyme Transport Disorders
Mucolipidosis 11(1-Cell N-Acetylglucosamine-1-
SEQ ID NO: 256 SEQ ID NO: 213
Disease) Phosphotransferase
NM_024312.5 NP_077288.2
(GNPTAB)(also referred (GNPTAB)
(GNPTAB)
to as GleNAc-l-
phosphotransferase)
Mucolipidosis III (Pseudo- Same as MLII
SEQ ID NO: 256 SEQ ID NO: 213
Hurler Polydystrophy)
F. Lysosomal Membrane
Transport Disorders
Cystinosis cystinosin (also referred
SEQ ID NO: 257 SEQ ID NO: 214
to as lysosomal cystine
NM_004937.3 NP_004928.2
transporter) (CTNS)
(CTNS) (CTNS)
Sialic acid storage disease solute carrier family 17
SEQ ID NO: 258 SEQ ID NO: 2015
(Salta Disease is a less member 5 (SLC17A5)
NM 012434.5 NP 036566.1
severe form) (also known as Sialin or
(SLC17A5) (SLC17A5)
Sialic Acid Transport
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Protein or SAISD, SD,
ISSD, NSD)
Infantile Sialic Acid solute carrier family 17
SEQ ID NO: 1258 SEQ ID NO: 215
Storage Disease (ISSD) member 5 (SLC17A5)
(also known as Slain' or
Sialic Acid Transport
Protein or SAISD, SD,
ISSD, NSD)
G. Other
CLN3 disease Battenin (also referred to
SEQ ID NO: 259 SEQ ID NO: 216
(including Batten Disease as CLN3
NM_000086.2 NP_000077.1
(Juvenile Neuronal Ceroid lysosomal/endosomal
(CLN3) (CLN3)
Lipofitscinosis) transmembrarte protein or
Batten) (CLN3)
Infantile Neuronal Palmitoyl-Protein
SEQ ID NO: 260 SEQ ID NO: 217
Ceroid Lipofuscinosis (or Thioesterase 1(PPT1
NM 000310.3 (PPT1) NP 000301.1
infantile Batten gene)
(P11)
disease)(CLN1 disease)
Mucolipidosis W mucolipin-1 (MCOLN1)
SEQ ID NO: 261 SEQ ID NO: 218
NM_020533.3
NP_065394.1(MCO
(MCOLN1)
LN1)
Prosaposin Deficiency Prosaposin (PSAP).
SEQ ID NO: 262 SEQ ID NO: 219
(associated with PSAP protein is the
PSAP (NM_002778.4) NP_002769.1
metachromatic precursor of four smaller
(PSAP)
leukodystrophy or PSAP proteins called saposin A,
mutation) B, C, and D)
1005281 Table 6B: Exemplary Lysosomal Storage Diseases (LSD) and proteins to
be expressed by
targeting vectors or rAAV vectors, and nucleic acid sequences encoding the
proteins.
Disease Enzyme defect
Nucleic acid sequence Protein sequence
Ehlers-Danlos PLOD! lysyl SEQ
ID NO: 263 SEQ ID NO: 220
Syndrome Type Hydroxylase (PLOD1) NM
000302.4 NP 000293.2
VI (pr000llagen-lysine,2-
oxoglutarate 5-dioxygenase
I)
Type la glycogen glucose6 phosphatase SEQ
ID NO: 264 SEQ ID NO: 221
storage disease catalytic subunit (G6PC)
NM_000151.4 (G6PC) NP_000142.2
(GSDIa)
(G6PC)
Type lb glycogen solute carrier family 37 SEQ
ID NO: 265 SEQ ID NO: 222
storage disease member 4 (also known as
NM_001467.6 (5LC37A4) NP_001458.1
(GSDIb) glucose 6-phosphate
(SLC37A4)
translocase protein or
G6PT1 or GSD1b)
(SLC37A4)
Congenital Disorders ciGlycosylation
ALG6-congenital ALG6 a1,3 SEQ
ID NO: 266 SEQ ID NO: 223
disorder of glucosyltransferase (ALG6) NM_013339.4
(ALG6) NP_037471.2
glycosylation
(ALG6)
(ALG6-CDG) (also
known as
congenital disorder
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of glycosylation
type Ic or CDG Ic)
congenital disorder ALG3
SEQ ID NO: 267 SEQ ID NO: 224
of glycosylation a1,3 NM
005787.6 (ALG3) NP 005778.1
type Id (CDG-Id) mannosyltransferase (ALG3)
(ALG3)
characterized by
abnormal N-
glycosylation
Congenital disorder MGAT2 (encodes
SEQ ID NO: 268 SEQ ID NO: 225
of glycosylation 2A N-acetylglucosaminyl-
NM 002408.4 NP 002399.1
(CDG2A or CDG transferase II (also referred (MGAT2)
(MGAT2)
Ha) to as alpha-1,6-mannosyl-
glycopmtein 2-beta-N-
acetylglucosaminyltransferas
e or GNT-II)
Type IIb congenital GCSI
SEQ ID NO: 269 SEQ ID NO: 226
disorder of a1,2-Glucosidase I (GDG2B NM_006302.3
(MOGS) NP 006293.2
glycosylation or GCS1) (also known as
(MOGS)
(CDGIIb) mannosyl-oligosaccharide
glucosidase or MOGS)
VII. Administration
1005291ln some embodiments, the technology described herein relates to methods
and compositions
for administering a rAAV vector or rAAV genome as disclosed herein to a
subject with a lysosomal
disease or disorder, e.g., to a subject with Pompe disease. In some
embodiments, the method
comprises administering a composition comprising a homogenous population of
rAAV vector
comprising a rAAV vector, where the rAAV vector is a AAV3 or AAV8 vector, or a
haploid rAAV
vector comprising at least one capsid protein from AAV3 or AAV8 as disclosed
herein. In some
embodiments, the subject is administered a cocktail of different rAAV vectors
as disclosed herein,
e.g., a compostion comprising a rAAV vector targeting or transduces liver
cells and a rAAV vector
that targets or transduces muscle cells. In an exemplary embodiment, a subject
is administered a
composition comprising cocktail of two or more different rAAV vectors as
disclosed herein, e.g., a
compostion comprising a AAV3 vector and AAV8 vector as disclosed herein, where
each AAV
vector comprises a nucleic acid encoding a GAA polypeptide operatively linked
to a LSP as disclosed
herein.
10053011n some embodiments, the subject is co-administered, for example at the
same time, or
subsequently, two or more different rAAV vectors as disclosed herein. For
exemplary purposes only,
a subject can be administered a compostion comprising a rAAV vector that
targets or transduces liver
cells and where the subject is also co-administered a composition comprising a
rAAV vector that
targets or transduces muscle cells. In an exemplary embodiment, a subject is
co-administered a
composition comprising a rAAV vector as disclosed herein, e.g., a compostion
comprising a AAV3
vector or variant thereof as disclosed herein, which comprises a nucleic acid
encoding a GAA
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polypeptide operatively linked to a LSP as disclosed herein, and where the
subject is also co-
administered a composition comprising a different rAAV vector as disclosed
herein, e.g., a
compostion comprising a AAVS vector or haploid AAV vector which comprises a
nucleic acid
encoding a GAA polypeptide operatively linked to a LSP as disclosed herein, or
alternatively, where
the LSP is replaced with a different promoter, e.g., a muscle specific
promoter.
1005311Dosages of the a rAAV vector or rAAV genome as disclosed herein to be
administered to a
subject depend upon the mode of administration, the severity of the Pompe
disease or other condition
to be treated and/or prevented, the individual subject's condition, the
particular virus vector or capsid,
and the nucleic acid to be delivered, and the like, and can be determined in a
routine manner.
Exemplary doses for achieving therapeutic effects are titers of at least about
105, 106, 101, 108, 109,
10w, 10", 1012, 10", 10'4, 1015transducing units, optionally about 108 to
about 10" transducing units.
1005321In a further embodiment, administration of rAAV vector or rAAV genome
as disclosed herein
to a subject results in production of a GAA protein with a circulatory half-
life of 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12
hours, 13 hours, 14 hours, 15
hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours,
23 hours, 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one
month, two months,
three months, four months or more.
1005331In an embodiment, the period of administration of a rAAV vector or rAAV
genome as
disclosed herein to a subject is for 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9 days,
days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9
weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8
months, 9 months,
10 months, 11 months, 12 months, or more. In a further embodiment, a period of
during which
administration is stopped is for 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9 days, 10
days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks,
7 weeks, 8 weeks, 9
weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8
months, 9 months,
10 months, 11 months, 12 months, or more.
1005341 In another embodiment, administration of a rAAV vector or rAAV genome
as disclosed
herein for the treatment of Pompe Disease results in an increase in weight by,
e.g., at least 0.5 pounds,
at least 1 pound, at least 1.5 pounds, at least 2 pounds, at least 2.5 pounds,
at least 3 pounds, at least
3.5 pounds, at least 4 pounds, at least 4.5 pounds, at least 5 pounds, at
least 5.5 pounds, at least 6
pounds, at least 6.5 pounds, at least 7 pounds, at least 7.5 pounds, at least
8 pounds, at least 8.5
pounds, at least 9 pounds, at least 9.5 pounds, at least 10 pounds, at least
10.5 pounds, at least 11
pounds, at least 11.5 pounds, at least 12 pounds, at least 12.5 pounds, at
least 13 pounds, at least 13.5
pounds, at least 14 pounds, at least 14.5 pounds, at least 15 pounds, at least
20 pounds, at least 25
pounds, at least 30 pounds, at least 50 pounds.
1005351 In another embodiment, an AAV GAA of any serotype, as disclosed herein
for the treatment
of Pompe Disease results in an increase in weight by, e.g., from 0.5 pounds to
50 pounds, from 0_5
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pounds to 30 pounds, from 0.5 pounds to 25 pounds, from 0.5 pounds to 20
pounds, from 0.5 pounds
to 15 pounds, from 0.5 pounds to ten pounds, from 0.5 pounds to 7.5 pounds,
from 0.5 pounds to 5
pounds, from 1 pound to 15 pounds, from 1 pound to 10 pounds, from 1 pound to
7.5 pounds, form 1
pound to 5 pounds, from 2 pounds to ten pounds, from 2 pounds to 7.5 pounds.
A Pharmaceutical Compositions
[001] The rAAV vectors as disclosed herein for use in the methods of
administration as disclosed
herein can be formulated in a pharmaceutical composition with a
pharmaceutically acceptable
excipient, i.e., one or more pharmaceutically acceptable carrier substances
and/or additives, e.g.,
buffers, carriers, excipients, stabilizers, etc. The pharmaceutical
composition may be provided in the
form of a kit. Pharmaceutical compositions comprising the rAAV vectors as
disclosed herein for use
in the methods of administration as disclosed herein and uses thereof are
known in the art.
[002] Accordingly, a further aspect of the invention provides a pharmaceutical
composition
comprising a rAAV vector as disclosed herein for use in the methods of
administration as disclosed
herein. Relative amounts of the active ingredient (e.g. a rAAV vectors aa
disclosed herein), a
pharmaceutically acceptable excipient, and/or any additional ingredients in a
pharmaceutical
composition in accordance with the present disclosure may vary, depending upon
the identity, size,
and/or condition of the subject being treated and further depending upon the
route by which the
composition is to be administered. For example, the composition may comprise
between 0.1 percent
and 99 percent (w/w) of the active ingredient. By way of example, the
composition may comprise
between 0.1 percent and 100 percent, e.g., between.5 and 50 percent, between 1-
30 percent, between
5- 80 percent, at least 80 percent (w/w) active ingredient.
[003] The pharmaceutical compositions can be formulated using one or more
excipients or diluents
to (1) increase stability; (2) increase cell transfection or transduction; (3)
permit the sustained or
delayed release of the payload; (4) alter the biodistribution (e.g., target
the viral particle to specific
tissues or cell types); (5) increase the translation of encoded protein; (6)
alter the release profile of
encoded protein and/or (7) allow for regulatable expression of the payload of
the invention. In some
embodiments, a pharmaceutically acceptable excipient may be at least 95
percent, at least 96 percent,
at least 97 percent, at least 98 percent, at least 99 percent, or 100 percent
pure. In some embodiments,
an excipient is approved for use for humans and for veterinary use. In some
embodiments, an
excipient may be approved by United States Food and Drug Administration. In
some embodiments,
an excipient may be of pharmaceutical grade. In some embodiments, an excipient
may meet the
standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia
(EP), the British
Pharmacopoeia, and/or the International Pharmacopoeia. Excipients, as used
herein, include, but are
not limited to, any and all solvents, dispersion media, diluents, or other
liquid vehicles, dispersion or
suspension aids, surface active agents, isotonic agents, thickening or
emulsifying agents,
preservatives, and the like, as suited to the particular dosage form desired.
Various excipients for
formulating pharmaceutical compositions and techniques for preparing the
composition are known in
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the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition,
A. R. Gennaro,
Lippincott, Williams and Wilkins, Baltimore, MD, 2006; 'incorporated herein by
reference in its
entirety). The use of a conventional excipient medium may be contemplated
within the scope of the
present disclosure, except insofar as any conventional excipient medium may be
incompatible with a
substance or its derivatives, such as by producing any undesirable biological
effect or otherwise
interacting in a deleterious manner with any other component(s) of the
pharmaceutical composition.
[004] The rAAV vectors as disclosed herein for use in the methods of
administration as disclosed
herein may be used in combination with one or more other therapeutic,
prophylactic, research or
diagnostic agents. By "in combination with," it is not intended to imply that
the agents must be
administered at the same time and/or formulated for delivery together,
although these methods of
delivery are within the scope of the present invention. Compositions can be
administered concurrently
with, prior to, or subsequent to, one or more other desired therapeutics or
medical procedures. In
some embodiments, the delivery of one treatment (e.g., gene therapy vectors)
is still occurring when
the delivery of the second (e.g., one or more therapeutic) begins, so that
there is overlap in terms of
administration. This is sometimes referred to herein as "simultaneous" or
"concurrent delivery." In
other embodiments, the delivery of one treatment ends before the delivery of
the other treatment
begins. In some embodiments of either case, the treatment is more effective
because of combined
administration. For example, the second treatment is more effective, e.g., an
equivalent effect is seen
with less of the second treatment, or the second treatment reduces symptoms to
a greater extent, than
would be seen if the second treatment were administered in the absence of the
first treatment, or the
analogous situation is seen with the first treatment. In some embodiments,
delivery is such that the
reduction in a symptom, or other parameter related to the disorder is greater
than what would be
observed with one treatment delivered in the absence of the other. The effect
of the two treatments
can be partially additive, wholly additive, or greater than additive. The
delivery can be such that an
effect of the first treatment delivered is still detectable when the second is
delivered. The composition
described herein and the at least one additional therapy can be administered
simultaneously, in the
same or in separate compositions, or sequentially. For sequential
administration, the gene therapy
vectors described herein can be administered first, and the one or more
therapeutic can be
administered second, or the order of administration can be reversed. The gene
therapy vectors and the
one or more therapeutic can be administered during periods of active disorder,
or during a period of
remission or less active disease. The gene therapy vectors can be administered
before another
treatment, concurrently with the treatment, post-treatment, or during
remission of the disorder.
[005] When administered in combination, the rAAV vectors as disclosed herein
for use in the
methods of administration as disclosed herein and the one or more therapeutic
(e.g., second or third
therapeutic), or all, can be administered in an amount or dose that is higher,
lower or the same as the
amount or dosage of each used individually, e.g., as a monotherapy. In certain
embodiments, the
administered amount or dosage of a rAAV vector as disclosed herein for use in
the methods of
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administration as disclosed herein and the one or more therapeutic (e.g.,
second or third agent), or all,
is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)
than the amount or dosage of
each used individually. In other embodiments, the amount or dosage of the rAAV
vector as disclosed
herein for use in the methods of administration as disclosed herein and the
one or more therapeutic
(e.g., second or third agent), or all, that results in a desired effect (e.g.,
treatment of a cardiovascular
disease or heart disease) is lower (e.g., at least 20%, at least 30%, at least
40%, or at least 50% lower)
than the amount or dosage of each individually required to achieve the same
therapeutic effect.
[006] In some embodiments, the methods of administration of a rAAV vector as
disclosed herein
can deliver a rAVV vector disclosed herein alone, or in combination with an
additional agent, for
example, an immune modulator as disclosed herein.
B. Immune Modulator:
[00536] In some embodiments, the methods and compositions using the AAV
vectors and AAV
genomes as described herein, for treating lysosomal disease, e.g., Pompe,
further comprises
administering an immune modulator. In some embodiments, the immune modulator
can be
administered at the time of rAAV vector administration, before rAAV vector
adminishation or, after
the rAAV vector administration.
[00537] In some embodiments, the immune modulator is an immunoglobulin
degrading enzyme such
as IdeS, IdeZ, IdeS/Z, Endo S, or, their functional variant. Non-limiting
examples of references of
such immunoglobulin degrading enzymes and their uses as described in US
7,666,582, US 8,133,483,
US 20180037962, US 20180023070, US 20170209550, US 8,889,128, W02010/057626,
US
9,707,279, US 8,323,908, US 20190345533, US 20190262434, and W02020/016318,
each of which
are incorporated in their entirety by reference.
[00538] In some embodiments, the immune modulator is Proteasome inhibitor. In
certain aspects, the
proteasome inhibitor is Bortezomib. In some aspects of the embodiment, the
immune modulator
comprises bortezomib and anti CD20 antibody, Riniximab. In other aspects of
the embodiment, the
immune modulator comprises bortezomib, Riniximab, methotrexate, and
intravenous gamma
globulin. Non-limiting examples of such references, disclosing proteasome
inhibitors and their
combination with Rituximab, methotrexate and intravenous gamma globulin, as
described in US
10,028,993, US 9,592,247, and, US 8,809,282, each of which are incorporated in
their entirety by
reference.
[00539] In alternative embodiments, the immune modulator is an inhibitor of
the NF-kB pathway. In
certain aspects of the embodiment, the immune modulator is Rapamycin or, a
functional variant. Non-
limiting examples of references disclosing rapaanycin and its use described in
US 10,071,114, US
20160067228, US 20160074531, US 20160074532, US 20190076458, US 10,046,064,
are
incorporated in their entirety. In other aspects of the embodiment, the immune
modulator is synthetic
nanocarriers comprising an immunosuppressant. Non limiting examples of
references of
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immunosuppresants, immunosuppressants coupled to synthetic nanocarriers,
synthetic nanocarriers
comprising rapamycin, and/or, toloregenic synthetic nanocarriers, their doses,
administration and use
as described in U520150320728, US 20180193482, US 20190142974, US 20150328333,
US20160243253, US 10,039,822, US 20190076522, US 20160022650, US 10,441,651,
US
10,420,835, US 20150320870, US 2014035636, US 10,434,088, US 10,335,395, US
20200069659,
US 10,357,483, US 20140335186, US 10,668,053, US 10,357,482, US 20160128986,
US
20160128987, US 20200038462, US 20200038463, each of which are incorporated in
their entirety
by reference.
1005401ln some embodiments, the immune modulator is synthetic nanocarriers
comprising rapamycin
(ImmTORTm nanoparticles) (ICishirnoto, et al., 2016, Nat Nanotechnol, 11(10):
890-899; Maldonado,
et al., 2015, PNAS, 112(2): E156-165), as disclosed in U520200038463, US
Patent 9,006,254 each of
which is incorporated herein in its entirety_ In some embodiments, the immune
modulator is an
engineered cell, e.g., an immune cell that has been modified using SQZ
technology as disclosed in
W02017192786, which is incorporated herein in its entirety by reference.
1005411 In some embodiments, the immune modulator is selected from the group
consisting of poly-
ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909,
CyaA, dSLIM,
GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,
Juvlinmune,
LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,
Montanide ISA
50V, Montanide ISA-51, OK-432, 0M-174, 0M-197-MP-EC, ONTAK, PEPTEL, vector
system,
PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like
particles, YF-17D,
VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon. In another
further
embodiment, the immunomodulator or adjuvant is poly-ICLC
1005421 In some embodiments, the immune modulator is a small molecule that
inhibit the innate
immune response in cells, such as chloroquine (a TLR signaling inhibitor) and
2-aminopurine (a PICR
inhibitor), can also be administered in combination with the composition
comprising at least one
rAAV as disclosed herein. Some non-limiting examples of commercially available
TLR-signaling
inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and
rapamycin (all
available for purchase from INVIVOGENTm). In addition, inhibitors of pattern
recognition receptors
(PRR) (which am involved in innate immunity signaling) such as 2-aminopurine,
13X795,
chloroquine, and H-89, can also be used in the compositions and methods
comprising at least one
rAAV vector as disclosed herein for in vivo protein expression as disclosed
herein.
10054311n some embodiments, a rAAV vector can also encode a negative
regulators of innate
immunity such as NLRX1. Accordingly, in some embodiments, a rAAV vector can
also optionally
encode one or more, or any combination of NLRX1, NS1, NS3/4A, or A46R.
Additionally, in some
embodiments, a composition comprising at least one rAAV vector as disclosed
herein can also
comprise a synthetic, modified-RNA encoding inhibitors of the innate immune
system to avoid the
innate immune response generated by the tissue or the subject.
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10054411n some embodiments, an immune modulator for use in the administration
methods as
disclosed herein is an immunosuppressive agent. As used herein, the term
"immunosuppressive drug
or agent" is intended to include pharmaceutical agents which inhibit or
interfere with normal immune
fumction. Examples of immunosuppressive agents suitable with the methods
disclosed herein include
agents that inhibit T-cell/B- cell costimulation pathways, such as agents that
interfere with the
coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in
U.S. Patent Pub. No
2002/0182211. In one embodiment, an immunosuppressive agent is cyclosporine A.
Other examples
include myophenylate mofetil, raparnicin, and anti- thymocyte globulin. In one
embodiment, the
immunosuppressive drug is administered in a composition comprising at least
one rAAV vector as
disclosed herein, or can be administered in a separate composition but
simultaneously with, or before
or after administration of a composition comprising at least one rAAV vector
according to the
methods of administration as disclosed herein. An immunosuppressive drug is
administered in a
formulation which is compatible with the route of administration and is
administered to a subject at a
dosage sufficient to achieve the desired therapeutic effect. In some
embodiments, the
immunosuppressive drug is administered transiently for a sufficient time to
induce tolerance to the
rAAV vector as disclosed herein,
1005451ln any embodiment of the methods and compositions as disclosed herein,
a subject being
administered a rAAV vector or rAAV genome as disclosed herein is also
administered an
immunosuppressive agent. Various methods are known to result in the
immunosuppression of an
immune response of a patient being administered AAV. Methods known in the art
include
administering to the patient an immunosuppressive agent, such as a proteasome
inhibitor. One such
proteasome inhibitor known in the art, for instance as disclosed in U.S.
Patent No. 9,169,492 and U.S.
Patent Application No. 15/796,137, both of which are incorporated herein by
reference, is bortezomib.
In some embodiments, an immunosuppressive agent can be an antibody, including
polyclonal,
monoclonal, scfv or other antibody derived molecule that is capable of
suppressing the immune
response, for instance, through the elimination or suppression of antibody
producing cells. In a
further embodiment, the immunosuppressive element can be a short hairpin RNA
(shRNA). In such
an embodiment, the coding region of the shRNA is included in the rAAV cassette
and is generally
located downstream, 3' of the poly-A tail. The shRNA can be targeted to reduce
or eliminate
expression of inununostimulatory agents, such as cytokines, growth factors
(including transforming
growth factors 01 and J32, TNF and others that are publicly known).
1005461The use of such immune modulating agents facilitates the ability to for
one to use multiple
dosing (e.g., multiple administration) over numerous months and/or years. This
permits using multiple
agents as discussed below, e.g., a rAAV vector encoding multiple genes, or
multiple administrations
to the subject.
1005471A/1 aspects of the compositions and methods of the technology disclosed
herein can be defined
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in any one or more of the following numbered paragraphs:
1. A recombinant adenovirus associated (AAV) vector comprising in its genome:
(a) 5' and 3' AAV
inverted terminal repeats (ITR) sequences, and (b) located between the 5' and
3' ITRs, a
heterologous nucleic acid sequence encoding an alpha-glucosidase (GAA)
polypeptide, wherein
the heterologous nucleic acid is operatively linked to a liver-specific
promoter.
2. The recombinant AAV vector of paragraph 1, wherein the heterologous
nucleic acid sequence
encoding the GAA polypeptide further comprises a signal peptide located 5' of
the nucleic acid
encoding the alpha-gluoosidase (GAA) polypeptide, or wherein the heterologous
nucleic acid
sequence encoding the GAA polypeptide further comprises a IGF2 targeting
peptide at the N-
terminus of GAA polypeptide.
3. The recombinant AAV vector of paragraph 1, wherein the heterologous
nucleic acid sequence
encoding the GAA polypeptide further comprises a IGF2 targeting peptide
located between the
secretory signal peptide and the alpha-glucosidase (GAA) polypeptide, or
wherein the
heterologous nucleic acid sequence encoding the GAA polypeptide further
comprises a IGF2
targeting peptide at the N-terminus of GAA polypeptide.
4. The recombinant AAV vector of any of paragraphs 1-3, wherein the AAV
genome comprises,
in the 5' to 3' direction:(a) a 5' ITR, (b) a liver-specific promoter
sequence, (c) an intron
sequence, (d) a nucleic acid encoding a secretory signal peptide, (e) a
nucleic acid encoding an
alpha-glucosidase (GAA) polypeptide, (f) a poly A sequence, and (g) a 3' ITR.
In some
embodiments, the intron sequence (c) is absent.
5. The recombinant AAV vector of any of paragraphs 1-3, wherein the AAV
genome comprises,
in the 5' to 3' direction:(a) a 5' ITR, (b) a liver-specific promoter
sequence, (c) an intron
sequence, (d) a nucleic acid encoding an IGF2 targeting peptide, (e) a nucleic
acid encoding an
alpha-glucosidase (GAA) polypeptide, (f) a poly A sequence, and (g) a 3' Mt In
some
embodiments, the intron sequence (c) is absent.
6. The recombinant AAV vector of any of paragraphs 1-6, wherein the AAV
genome comprises,
in the 5' to 3' direction:(a) a 5' ITR, (b) a liver-specific promoter
sequence, (c) an intron
sequence, (d) a nucleic acid encoding a secretory signal peptide, (e) a
nucleic acid encoding an
IGF2 targeting peptide, (f) a nucleic acid encoding an alpha-glucosidase (GAA)
polypeptide,
(g) a poly A sequence, and (h) a 3' ITR.. In some embodiments, the intron
sequence is absent.
7. The recombinant AAV vector of any of paragraphs 1-6, wherein the
secretory signal peptide is
selected from an AAT signal peptide, a fibronectin signal peptide (FN1), a GAA
signal peptide,
or an active fragment thereof having secretory signal activity. For example,
the nucleic acid
encoding a secretory signal peptide, can be selected from an AAT signal
peptide (e.g., SEQ ID
NO: 17), a fibronectin signal peptide (FN1) (e.g., SEQ ID NO: 18-21), a
cognate GAA signal
peptide (SEQ ID NO: 175), an hIGF2 signal peptide (e.g., SEQ ID NO: 22), a
IgG1 leader
peptide (SEQ ID NO: 177), wtIL2 leader peptide (SEQ ID NO: 179), mutant IL2
leader peptide
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(SEQ ID NO: 181) or an active fragment thereof having secretory signal
activity, e.g., a nucleic
acid encoding an amino acid sequence that has at least about 75%, or 80%, or
85%, or 90%, or
95%, or 98%, or 99% sequence identity to SEQ ID NOs: 17-22, 175, 177, 179 or
181.
8. The recombinant AAV vector of any of paragraphs 1-7, wherein the IGF2
targeting peptide
binds human cation-independent mannose-6-phosphate receptor (CI-MPR) or the
IGF2
receptor.
9. The recombinant AAV vector of any of paragraphs 1-5, wherein the IGF2
targeting peptide
comprises SEQ ID NO: 5 or comprises at least one amino modification in SEQ ID
NO: 5 that
does not affect the binding to the CI-MPR receptor, or that reduces its
binding to one or more
IGF binding proteins (IGFBPs, such as IGFBP1-6).
10. The recombinant AAV vector of any of paragraphs 1-9, wherein the at
least one amino
modification in SEQ ID NO: 5 is a V43M amino acid modification (SEQ ID NO: 8
or SEQ ID
NO: 9) or A2-7 (SEQ ID NO: 6) or A1-7 (SEQ ID NO: 7).
11. The recombinant AAV vector of any of paragraphs 1-10, wherein the liver-
specific promoter is
a promoter of any of the sequence listed in Table 4 herein, or Table 4A or 4B
provisional
application 62,937,556, filed on November 19, 2019, or a functional variant
thereof or
functional fragment thereof
12. The recombinant AAV vector of any of paragraphs 1-10, wherein the liver-
specific promoter
comprises CRM_5P0412 (SEQ ID NO: 86) or 5P0412 (SEQ ID NO: 91) or a functional
variant
or functional fragment thereof having at least 60% activity to SEQ ID NO: 86
or SEQ ID NO:
91,
13. The recombinant AAV vector of any of paragraphs 1-12, wherein the liver
specific promoter is
selected from any of (i) SP0422 (SEQ ID NO: 92) or a functional variant or
functional
fragment thereof having at least 60% activity to SEQ ID NO: 92; (ii)
CRM_5P0239 (SEQ ID
NO: 87) or 5P0239 (SEQ ID NO: 93) or 5P0238-UTR (SEQ ID NO: 147) or a
functional
variant or functional fragment thereof having at least 60% activity to SEQ ID
NO: 87, SEQ ID
NO: 93 or SEQ ID NO: 147; (iii) CRM_5P0265 (SP0131 A 1) (SEQ ID NO: 88) or
5P0265
(LVR_SP0131_Al) (SEQ ID NO: 94) or SP0265-UTR (SEQ ID NO: 146) or a functional
variant or functional fragment thereof having at least 60% activity to SEQ ID
NO: 88, SEQ ID
NO: 94 or SEQ ID NO: 146; (iv) CRM_SP0240 (SEQ ID NO: 89) or 5P0240 (SEQ ID
NO: 95)
or 5P0240-UTR (SEQ ID NO: 148) or a functional variant or functional fragment
thereof
having at least 60% activity to SEQ ID NO: 89, SEQ ID NO: 95 or SEQ ID NO:
148; (v)
CRM SP0246 (SEQ ID NO: 90) or SP0246 (SEQ ID NO: 96) or 5P0246-UTR (SEQ ID NO:
149) or a functional variant or functional fragment thereof having at least
60% activity to SEQ
ID NO: 90, SEQ ID NO: 96 or SEQ ID NO: 149.
14. The recombinant AAV vector of any of paragraphs 1-13, wherein the
nucleic acid sequence
encodes a wild-type GAA polypeptide or a modified GAA polypeptide.
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15. The recombinant AAV vector of any of paragraphs 1-14, wherein the
nucleic acid sequence
encoding the GAA polypeptide is the human GAA gene or a human codon optimized
GAA
gene (coGAA) or a modified GAA nucleic acid sequence.
16. The recombinant AAV vector of any of paragraphs 1-15, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized for enhanced expression in
viva
17. The recombinant AAV vector of any of paragraphs 1-16, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce CpG islands.
18. The recombinant AAV vector of any of paragraphs 1-17, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce the innate immune
response or to
reduce CpG islands, or to reduce the innate immune response and reduce the
innate immune
response.
19. The recombinant AAV vector of any of paragraphs 1-18, wherein the
nucleic acid sequence
encodes a GAA polypeptide which comprises at least one, at least 2 or at least
all three amino
acid modifications selected from; H201L, H199R or R233H of SEQ ID NO: 10.
20. The recombinant AAV vector of any of paragraphs 1-19, wherein the
encoded polypeptide
further comprising a spacer comprising a nucleotide sequence for at least 1
amino acids located
amino-terminal to the GALA polypeptide, and C-terminal to the IGF2 targeting
peptide.
21. The recombinant AAV vector of any of paragraphs 1-20, further
comprising a nucleic acid
encoding a spacer of at least 1 amino acids located between the nucleic acid
encoding the IGF2
targeting peptide and the nucleic acid encoding the GAA polypeptide.
22. The recombinant AAV vector of any of paragraphs 1-21, further
comprising at least one polyA
sequence located 3' of the nucleic acid encoding the GAA gene and 5' of the 3'
ITR sequence.
23. The recombinant AAV vector of any of paragraphs 1-22, wherein the
heterologous nucleic acid
sequence further comprises at collagen stability (CS) sequence located 3' of
the nucleic acid
encoding the GAA polypeptide and 5' of the 3' ITR sequence.
24. The recombinant AAV vector of any of paragraphs 1-23, further
comprising a nucleic acid
encoding a collagen stability (CS) sequence located between the nucleic acid
encoding the GAA
polypeptide and the poly A sequence
25. The recombinant AAV vector of any of paragraphs 1-24, further
comprising an intron sequence
located 5' of the sequence encoding the secretory signal peptide, and 3' of
the promoter.
26. The recombinant AAV vector of any of paragraphs 1-25, wherein the
intron sequence
comprises a MVM sequence, SV40 sequence or a HEB2 sequence, wherein the MVM
sequence
comprises the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid
sequence at least
about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to
SEQ ID NO:
13, and the HI3132 sequence comprises the nucleic acid sequence of SEQ ID NO:
14, or a
nucleic acid sequence at least about 75%, or 80%, or 85%, or 90%, or 95%, or
98%, or 99%
sequence identity to SEQ ID NO: 14.
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27. The recombinant AAV vector of any of paragraphs 1-26, wherein the ITR
comprises an
insertion, deletion or substitution.
28. The recombinant AAV vector of any of paragraphs 1-27, wherein one or
more CpG islands in
the ITR are removed.
29. The recombinant AAV vector of any of paragraphs 1-28, wherein:
a. the heterologous nucleic acid sequence encodes
a secretory signal peptide selected
from:
1. a fibronectin signal peptide (FM!) or an active fragment thereof having
secretory
signal activity (e.g., a FN1 signal peptide has the sequence of any of SEQ ID
NO: 18-21,
or an amino acid sequence at having at least about 75%, or 800%, or 85%, or
90%, or 95%,
or 98%, or 99% sequence identity to any of SEQ ID NOs: 18-21),
2. an AAT signal peptide (es., SEQ ID NO: 17), or an amino acid sequence at
having at
least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to
any of SEQ ID NO: 17;
3. an hIGF2 signal peptide (e.g., SEQ ID NO: 22), or an amino acid sequence at
having
at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to
any of SEQ ID NO: 22;
4. a IgG1 leader peptide (SEQ ID NO: 177), or an amino acid sequence at
having at
least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to
any of SEQ ID NO: 177;
5. a wtIL2 leader peptide (SEQ ID NO: 179), or an amino acid sequence at
having at
least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to
any of SEQ ID NO: 179;
6. a mutant IL2 leader peptide (SEQ ID NO: 181) or an amino acid sequence at
having
at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to
any of SEQ ID NO: 181, and
b. the heterologous nucleic acid sequence encodes a
GAA polypeptide is selected from any
of the group consisting of: SEQ ID NO: 11, SEQ ID NO: 72 or SEQ ID NO: 182 or
a
nucleic acid sequence having at least 60%, or 70%, or 80%, 85% or 90% or 95%,
or
98%, or 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 72, or SEQ ID NO:
182, and optionally, the nucleic acid sequence encodes a GAA polypeptide with
at least
one, at least 2 or at least all three amino acid modifications selected from;
H201L,
H199R or R233H of SEQ ID NO: 10.
30. The recombinant AAV vector of paragraph 29, wherein the heterologous
nucleic acid also
encodes a IGF2 targeting peptide selected from any of: SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or a IGF2 peptide having at least about
75%, or 80%,
or 85%, or 90%, or 95%, 01 98%, 01 99% sequence identity to SEQ ID NOs: 5-9.
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31. The recombinant AAV vector of any of paragraphs 1-30, wherein the
encoded secretory signal
peptide is AAT signal peptide or an active fragment thereof having secretory
signal activity,
(e.g., a AAT signal peptide has the sequence of SEQ ID NO: 17, or an amino
acid sequence at
having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence identity
to SEQ ID NO: 17), and the heterologous nucleic acid sequence encodes a IGF2
targeting
peptide selected from any of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8 or
SEQ ID NO: 9, or a IGF2 peptide having at least about 75%, or 80%, or 85%, or
90%, or 95%,
or 98%, or 99% sequence identity to SEQ ID NOs: 5-9.
32. The recombinant AAV vector of any of paragraphs 1-31, wherein the IGF2
targeting peptide is
SEQ ID NO: 8 or SEQ ID NO: 9, or a IGF2 peptide having at least about 75%, or
80%, or 85%,
or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 8 or 9.
33. The recombinant AAV vector of any of paragraphs 1-32, wherein the
recombinant AAV vector
is selected from any of. a chimeric AAV vector, a haploid AAV vector, a hybrid
AAV vector, a
polyploid AAV vector, a rational haploid vector, a mosaic AAV vector, a
chemically modified
AAV vector, or a AAV vector from any AAV serotypes, e.g., any serotype listed
in Table 1 of
disclosed in International Applications W02020/102645, and W02020/102667.
34. The recombinant AAV vector of any of paragraphs 1-33, wherein the
recombinant AAV vector
comprises a capsid protein selected from any AAV serotype in the group
consisting of those
listed in Table 1 of U.S. provisional application 62,937,556, filed on
November 19, 2019, or in
International Applications W02020/102645, and W02020/102667, or and any
combination
thereof
35. The recombinant AAV vector of any of paragraphs 1-34, wherein the AAV
vector is selected
from a serotype from the group consisting of: a AAV3 vector, a AAVXL32 vector,
a
AAVXL32.1 vector, a AAV8 vector, or a haploid AAV8 vector comprising at least
one AAV8
capsid protein.
36. The recombinant AAV vector of any of paragraphs 1-35, wherein the AAV3b
serotype
comprises one or mutations in a capsid protein selected from any of: 265D,
549A, Q263Y
37. The recombinant AAV vector of any of paragraphs 1-36, wherein the AAV3b
serotype is
selected from any of AAV3b265D, AAV3b265D549A, AAV3b549A or AAV3bQ263Y, or
AAV3bSASTG.
38. A recombinant adenovirus associated (AAV) vector comprising in its
genome:
a. 5' and 3' AAV inverted terminal repeats (ITR) sequences, and
b. located between the 5' and 3' ITRs, a heterologous nucleic acid sequence
encoding an
alpha-glucosidase (GAA) polypeptide, wherein the heterologous nucleic acid is
operatively linked to a liver specific promoter, and
wherein the recombinant AAV vector comprises a capsid protein of the AAV8
serotype or
AAV3b serotype.
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39. The recombinant AAV vector of paragraph 38, wherein the GAA polypeptide
further comprises
a secretory signal peptide located at the N-terminal of the GAA polypeptide.
40. The recombinant AAV vector of paragraph 38-39, wherein the heterologous
nucleic acid
sequence encoding the GAA polypeptide further comprises a IGF2 targeting
peptide located N-
terminal of the GAA polypeptide, or located between the secretory signal
peptide and the an
alpha-glucosidase (GALA) polypeptide.
41. The recombinant AAV vector of paragraph 38, wherein the AAV genome
comprises, in the 5'
to 3' direction: a 5' ITR, a liver specific promoter sequence, a nucleic acid
encoding an alpha-
glucosidase (GAA) polypeptide, a poly A sequence, and a 3' ITR.
42. The recombinant AAV vector of paragraph 38, wherein the AAV genome
comprises, in the 5'
to 3' direction: a 5' ITR, a liver specific promoter sequence, a nucleic acid
encoding a secretory
signal peptide, a nucleic acid encoding an alpha-glucosidase (GAA)
polypeptide, a poly A
sequence, and a 3' ITR.
43. The recombinant AAV vector of paragraph 38, wherein the AAV genome
comprises, in the 5'
to 3' direction: a 5' ITR, a liver specific promoter sequence, an intron
sequence, a nucleic acid
encoding a secretory signal peptide, a nucleic acid encoding an alpha-
glucosidase (GAA)
polypeptide, a poly A sequence, and a 3' ITR.
44. The recombinant AAV vector of paragraph 38, wherein the AAV genome
comprises, in the 5'
to 3' direction: a 5' ITR, a liver specific promoter sequence, an intron
sequence, a nucleic acid
encoding a secretory signal peptide, a nucleic acid encoding a IGF2 targeting
peptide, a nucleic
acid encoding an alpha-glucosidase (GAA) polypeptide, a poly A sequence, and a
3' ITR.
45. The recombinant AAV vector of paragraph 38, wherein the AAV genome
comprises, in the 5'
to 3' direction: a 5' ITR, a liver specific promoter sequence, an intron
sequence, a nucleic acid
encoding a secretory signal peptide, a nucleic acid encoding a IGF2 targeting
peptide, a nucleic
acid encoding an alpha-glucosidase (GAA) polypeptide, a 3' ITR sequence, a
poly A sequence,
and a 3' ITR.
46. The recombinant AAV vector of any of paragraphs 34-37, wherein the
secretory signal peptide
is selected from nucleic acid encoding a secretory signal peptide, can be
selected from an AAT
signal peptide (e.g., SEQ ID NO: 17), a fibronectin signal peptide (FN1)
(e.g., SEQ ID NO: 18-
21), a cognate GAA signal peptide (SEQ ID NO: 175), an hIGF2 signal peptide
(e.g., SEQ ID
NO: 22), a IgG1 leader peptide (SEQ ID NO: 177), wtIL2 leader peptide (SEQ ID
NO: 179),
mutant IL2 leader peptide (SEQ ID NO: 181) or an active fragment thereof
having secretory
signal activity, e.g., a nucleic acid encoding an amino acid sequence that has
at least about 75%,
or 80%, or 85%, or 90%, 01 95%, 01 98%, or 99% sequence identity to SEQ ID
NOs: 17-22,
175, 177, 179 or 181.
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47. The recombinant AAV vector of any of paragraphs 38-46, wherein the IGF2
targeting peptide
binds human cation-independent mannose-6-phosphate receptor (CI-MPR) or the
IGF2
receptor.
48. The recombinant AAV vector of any of paragraphs 38-47, wherein the IGF2
targeting peptide
comprises SEQ ID NO: 5 or comprises at least one amino modification in SEQ ID
NO: 5 that
does not affect binding to the IGF2 receptor or reduces its binding to one or
more of IGF
Binding proteins IGFBP1-6.
49. The recombinant AAV vector of paragraph 48, wherein the at least one
amino modification in
SEQ ID NO: 5 is a V43M amino acid modification (SEQ ID NO: 8 or SEQ ID NO: 9)
or A2-7
(SEQ ID NO: 6) or A1-7 (SEQ ID NO: 7).
50. The recombinant AAV vector of any of paragraphs 38-49, wherein the
liver specific promoter is
selected from any of the sequences listed in Table 4, or Table 4A or 4B
provisional application
62,937,556, filed on November 19, 2019, or a functional variant or functional
fragment thereof
51. The recombinant AAV vector of any of paragraphs 38-50, wherein the
liver specific promoter is
selected from any of CRNI_SP0412 (SEQ ID NO: 86) or 5P0412 (SEQ ID NO: 91) or
a
functional variant or functional fragment thereof having at least 60% activity
to SEQ ID NO: 86
or SEQ ID NO: 91.
52. The recombinant AAV vector of any of paragraphs 38-50, wherein the
liver specific promoter is
selected from any of (i) 5P0422 (SEQ ID NO: 92) or a functional variant or
functional
fragment thereof having at least 60% activity to SEQ ID NO: 92; (ii) CRM
5P0239 (SEQ ID
NO: 87) or SP0239 (SEQ ID NO: 93) or 5P0238-UTR (SEQ ID NO: 147) or a
functional
variant or functional fragment thereof having at least 60% activity to SEQ ID
NO: 87, SEQ ID
NO: 93 or SEQ ID NO: 147; (iii) CRM SP0265 (SP0131 A 1) (SEQ ID NO: 88) or
5P0265
(LVR SP0131_Al) (SEQ ID NO: 94) or 5P0265-UTR (SEQ ID NO: 146) or a functional
variant or functional fragment thereof having at least 60% activity to SEQ ID
NO: 88, SEQ ID
NO: 94 or SEQ ID NO: 146; (iv) CRNI_5P0240 (SEQ ID NO: 89) or SP0240 (SEQ ID
NO: 95)
or 5P0240-UTR (SEQ ID NO: 148) or a functional variant or functional fragment
thereof
having at least 60% activity to SEQ ID NO: 89, SEQ ID NO: 95 or SEQ ID NO:
148; (v)
CRM_SP0246 (SEQ ID NO: 90) or SP0246 (SEQ ID NO: 96) or SP0246-UTR (SEQ ID NO:
149) or a functional variant or functional fragment thereof having at least
60% activity to SEQ
ID NO: 90, SEQ ID NO: 96 or SEQ ID NO: 149.
51 The recombinant AAV vector of paragraphs 38-52, wherein
the nucleic acid sequence encodes
a wild-type GAA polypeptide or a modified GAA polypeptide.
54. The recombinant AAV vector of any of paragraphs 38-53,
wherein the nucleic acid sequence
encoding the GAA polypeptide is the human GAA gene or a human codon optimized
GAA
gene (coGAA) or a modified GAA nucleic acid sequence.
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55. The recombinant AAV vector of any of paragraphs 38-54, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized for enhanced expression in
viva
56. The recombinant AAV vector of any of paragraphs 38-55, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce CpG islands.
57. The recombinant AAV vector of any of paragraphs 38-56, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce the innate immune
response, or
reduce the CpG islands, or reduce the innate immune response and reduce the
CpG islands.
58. The recombinant AAV vector of any of paragraphs 38-57, wherein the
nucleic acid sequence
encoding the GAA polypeptide encodes a GAA polypeptide comprising at least
one, at least 2
or at least all three amino acid modifications selected from; H201L, H199R or
R233H of SEQ
ID NO: 10.
59. The recombinant AAV vector any of paragraphs 38-58, wherein the intron
sequence is selected
from any of the MVM, HBB2 or SV40 intron sequence, wherein the MVM sequence
comprises
the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence at
least about 75%, or
80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to SEQ ID NO:
13, and the
HIBB2 sequence comprises the nucleic acid sequence of SEQ ID NO: 14, or a
nucleic acid
sequence at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence
identity to SEQ ID NO: 14.
60. The recombinant AAV vector any of paragraphs 38-59, wherein the ITR
comprises an insertion,
deletion or substitution.
61. The recombinant AAV vector of paragraph 60, wherein one or more CpG
islands in the ITR are
removed.
62. The recombinant AAV vector of any of paragraphs 38-61, wherein the 3'
ITR comprises or
consist of SEQ ID NO: 442, or a nucleotide sequence that is at least 80%
identical, e.g., at least
85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to SEQ ID NO: 442, and
the 5'
ITR comprises, or consists of SEQ ID NO: 441 or a nucleotide sequence that is
at least 80%
identical, e.g., at least 35%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5%
identical to SEQ ID
NO: 441.
63. The recombinant AAV vector of any of paragraphs 38-61, wherein the 3'
ITR comprises or
consist of SEQ ID NO: 165, or a nucleotide sequence that is at least 80%
identical, e.g., at least
85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to SEQ ID NO: 165, and
the 5'
ITR comprises, or consists of SEQ ID NO: 161 or a nucleotide sequence that is
at least 80%
identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5%
identical to SEQ ID
NO: 161.
64. The recombinant AAV vector of any of paragraphs 38-63, wherein the
secretory signal peptide
is a fibronectin signal peptide (FN1) or an active fragment thereof having
secretory signal
activity, (e.g., a FN1 signal peptide has the sequence of any of SEQ ID NO: 18-
21, or an amino
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acid sequence at having at least about 75%, or 80%, or 85%, or 90%, or 95%, or
98%, 01 99%
sequence identity to any of SEQ ID NOs: 18-21), and the heterologous nucleic
acid sequence
encodes a IGF2 targeting peptide selected from any of: SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or a IGF2 peptide having at least about
75%, or 80%,
or 85%, or 90%, or 95%, 01 98%, or 99% sequence identity to SEQ ID NOs: 5-9.
65. The recombinant AAV vector of any of paragraphs 38-64, wherein the
encoded secretory signal
peptide is AAT signal peptide or an active fragment thereof having secretory
signal activity,
(e.g., a AAT signal peptide has the sequence of SEQ ID NO: 17, or an amino
acid sequence at
having at least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%
sequence identity
to SEQ ID NO: 17), and the heterologous nucleic acid sequence encodes a IGF2
targeting
peptide selected from any of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8 or
SEQ ID NO: 9, or a IGF2 peptide having at least about 75%, or 80%, or 85%, or
90%, or 95%,
or 98%, or 99% sequence identity to SEQ ID NOs: 5-9.
66. The recombinant AAV vector of any of paragraphs 38-65, wherein the IGF2
targeting peptide is
SEQ ID NO: 8 or SEQ ID NO: 9, or a IGF2 peptide having at least about 75%, or
80%, or 85%,
or 90%, or 95%, or 98%, 01 99% sequence identity to SEQ ID NO: 8 or 9,
67. The recombinant AAV vector of any of paragraphs 38-66, wherein the
liver specific promoter is
CRM SP0412 (SEQ ID NO: 86) or SP0412 (SEQ ID NO: 91) or a functional variant
or
functional fragment thereof having at least 60% activity to SEQ ID NO: 86 or
SEQ ID NO: 91.
68. The recombinant AAV vector of any of paragraphs 38-66, wherein the
liver specific promoter is
5P0422 (SEQ ID NO: 92) or a functional variant or functional fragment thereof
having at least
60% activity to SEQ ID NO: 92.
69. The recombinant AAV vector of any of paragraphs 38-66, wherein the
liver specific promoter is
CRM SP0239 (SEQ ID NO: 87) or SP0239 (SEQ ID NO: 93) or 5P0238-UTR (SEQ ID NO:
147) or a functional variant or functional fragment thereof having at least
60% activity to SEQ
ID NO: 87, SEQ ID NO: 93 or SEQ ID NO: 147.
70. The recombinant AAV vector of any of paragraphs 38-66, wherein the
liver specific promoter is
CRM SP0265 (SP0131 Al) (SEQ ID NO: 88) or 5P0265 (LVR 5P0131 Al) (SEQ ID NO:
94) or 5P0265-UTR (SEQ ID NO: 146) or a functional variant or functional
fragment thereof
having at least 60% activity to SEQ ID NO: 88, SEQ ID NO: 94 or SEQ ID NO:
146.
71. The recombinant AAV vector of any of paragraphs 38-66, wherein the
liver specific promoter is
CRM SP0240 (SEQ ID NO: 89) or SP0240 (SEQ ID NO: 95) or 5P0240-UTR (SEQ ID NO:
148) or a functional variant or functional fragment thereof having at least
60% activity to SEQ
ID NO: 89, SEQ ID NO: 95 or SEQ ID NO: 148.
72. The recombinant AAV vector of any paragraphs 38-66, wherein the liver
specific promoter is
CRM_5P0246 (SEQ ID NO: 90) or SP0246 (SEQ ID NO: 96) or 5P0246-UTR (SEQ ID NO:
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149) or a functional variant or functional fragment thereof having at least
60% activity to SEQ
ID NO: 90, SEQ ID NO: 96 or SEQ ID NO: 149
71 A pharmaceutical composition comprising the recombinant
AAV vector of any one of the
previous paragraphs in a pharmaceutically acceptable carrier.
74. A nucleic acid sequence comprising:
a liver specific promoter operatively linked to a heterologous nucleic acid
sequence, the
heterologous nucleic acid sequence encoding a GAA polypeptide, wherein the
liver specific
promoter is selected from any one of the sequences disclosed in Table 4, or
Table 4A or 4B of
U.S. provisional application 62,937,556, filed on November 19, 2019, or a
functional variant or
functional fragment thereof.
75. The nucleic acid sequence of paragraph 74, wherein the
heterologous nucleic acid sequence
comprises in the following order: a nucleic acid encoding a secretory signal
peptide, and a
nucleic acid encoding a GAA polypeptide.
76. The nucleic acid sequence of paragraph 74, wherein the
heterologous nucleic acid sequence
comprises in the following order: a nucleic acid encoding a secretory signal
peptide, a nucleic
acid encoding a IGF2 targeting peptide and a nucleic acid encoding a GAA
polypeptide.
77. A nucleic acid sequence for a recombinant adenovirus
associated (rAAV) vector genome
comprising:
a. 5' and 3' AAV inverted terminal repeats (ITR) nucleic acid sequences, and
b. located between the 5' and 3' ITR sequence, a heterologous nucleic acid
sequence
encoding a polypeptide comprising an alpha-glucosidase (GAA) polypeptide,
wherein the
heterologous nucleic acid is operatively linked to a liver-specific promoter,
wherein the liver
specific promoter is selected from any one of the sequences disclosed in Table
4, or Table 4A or
4B of U.S. provisional application 62,937,556, filed on November 19, 2019, or
a functional
variant or functional fragment thereof
78. A nucleic acid sequence of paragraph 75, wherein the
nucleic acid sequence encodes a fusion
polypeptide comprising a secretory signal and an alpha-glucosidase (GAA)
polypeptide.
79. A nucleic acid sequence of paragraph 75, wherein the
nucleic acid sequence encodes a fusion
polypeptide comprising a secretory signal, an IGF2 targeting peptide and an
alpha-glucosidase
(GAA) polypeptide.
80. A nucleic acid sequence comprising:
a liver specific promoter operatively linked to a heterologous nucleic acid
sequence
comprising, a nucleic acid encoding a GAA polypeptide, wherein the liver
specific promoter is
selected from any of
i. CRI1,4 SP0412 (SEQ ID NO: 86) or 5P0412 (SEQ ID NO: 91) or a
functional variant or
functional fragment thereof having at least 60% activity to SEQ ID NO: 86 or
SEQ ID
NO: 91,
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5P0422 (SEQ ID NO: 92) or a functional variant or functional fragment thereof
having at
least 60% activity to SEQ ID NO: 92,
CRM SP0239 (SEQ ID NO: 87) or 5P0239 (SEQ ID NO: 93) or 5P0238-UTR (SEQ ID
NO: 147) or a functional variant or functional fragment thereof having at
least 60%
activity to SEQ ID NO: 87, SEQ ID NO: 93 or SEQ ID NO: 147;
iv. CRNI SP0265 (SP0131_A1) (SEQ ID NO: 88) or 5P0265 (LVR_SP0131_A1) (SEQ
ID
NO: 94) or 5P0265-UTR (SEQ ID NO: 146) or a functional variant or functional
fragment thereof having at least 60% activity to SEQ ID NO: 88, SEQ ID NO: 94
or SEQ
ID NO: 146;
v. CRNI SP0240 (SEQ ID NO: 89) or 5P0240 (SEQ ID NO: 95) or SP0240-UTR (SEQ
ID
NO: 148) or a functional variant or functional fragment thereof having at
least 60%
activity to SEQ ID NO: 89, SEQ ID NO: 95 or SEQ ID NO: 148; or
vi. CRNI SP0246 (SEQ ID NO: 90) or 5P0246 (SEQ ID NO: 96) or SP0246-UTR
(SEQ ID
NO: 149) or a functional variant or functional fragment thereof having at
least 60%
activity to SEQ ID NO: 90, SEQ ID NO: 96 or SEQ ID NO: 149.
81. A nucleic acid sequence of paragraph 80, wherein the heterologous
nucleic acid sequence
comprises in the following order: a nucleic acid encoding a secretory signal
and nucleic acid
encoding an alpha-glucosidase (GAA) polypeptide.
82. A nucleic acid sequence of paragraph 75, wherein the heterologous
nucleic acid sequence
comprises in the following order: a nucleic acid encoding a secretory signal,
a nucleic acid
sequence encoding an IGF2 targeting peptide and nucleic acid encoding an alpha-
glucosidase
(GAA) polypeptide.
83. A nucleic acid sequence for a recombinant adenovirus associated (rAAV)
vector genome
comprising: (a) 5' and 3' AAV inverted terminal repeats (ITR) nucleic acid
sequences, and (b)
located between the 5' and 3' ITR sequence, a heterologous nucleic acid
sequence encoding an
alpha-glucosidase (GAA) polypeptide, or encoding a fusion polypeptide
comprising a secretory
signal peptide and an alpha-glucosidase (GAA) polypeptide, wherein the
heterologous nucleic
acid is operatively linked to a liver-specific promoter, wherein the liver
specific promoter is
selected from any one of:
i. CRM SP0412 (SEQ ID NO: 86) or SP0412 (SEQ
ID NO: 91) or a functional
variant or functional fragment thereof having at least 60% activity to SEQ ID
NO:
86 or SEQ ID NO: 91,
5P0422 (SEQ ID NO: 92) or a functional variant or functional fragment thereof
having at least 60% activity to SEQ ID NO: 92,
CRM SP0239 (SEQ ID NO: 87) or 5P0239 (SEQ ID NO: 93) or 5P0238-UTR
(SEQ ID NO: 147) or a functional variant or functional fragment thereof having
at
least 60% activity to SEQ ID NO: 87, SEQ ID NO: 93 or SEQ ID NO: 147;
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iv. CRM SP0265 (SP013 LAD (SEQ ID NO: 88) or 5P0265 (LVR_SP0131_Al)
(SEQ ID NO: 94) or SP0265-UTR (SEQ ID NO: 146) or a functional variant or
functional fragment thereof having at least 60% activity to SEQ ID NO: 88, SEQ
ID NO: 94 or SEQ ID NO: 146;
v. CRM SP0240 (SEQ ID NO: 89) or 5P0240 (SEQ ID NO: 95) or 5P0240-UTR
(SEQ ID NO: 148) or a functional variant or functional fragment thereof having
at
least 60% activity to SEQ ID NO: 89, SEQ ID NO: 95 or SEQ ID NO: 148; or
vi. CRM SP0246 (SEQ ID NO: 90) or 5130246 (SEQ ID NO: 96) or 5P0246-UTR
(SEQ ID NO: 149) or a functional variant or functional fragment thereof having
at
least 60% activity to SEQ ID NO: 90, SEQ ID NO: 96 or SEQ ID NO: 149.
84. The nucleic acid sequence of any of paragraphs 74-83, wherein the
heterologous nucleic acid
sequence encoding the GAA polypeptide, or the fusion polypeptide further
comprises a IGF2
targeting peptide located between the secretory signal peptide and the an
alpha-glucosidase
(GAA) polypeptide.
85. The nucleic acid sequence of any of paragraphs 74-84, wherein the
nucleic acid encoding the
secretory signal is selected from any of SEQ ID NO: 17, 22-26, 175, 177, 179
or 18 lor a
nucleic acid sequence at least about 75%, or 800/c, or 85%, or 90%, or 95%, or
98%, or 99%
sequence identity to any of SEQ ID NOs: 17 or 22-26, or 175, 177, 179 or 181.
86. The nucleic acid sequence of any of paragraphs 74-85, wherein the
nucleic acid encoding the
IGF2 targeting peptide is selected from any of SEQ ID NO: 2 (IGF2-A2-7), SEQ
ID NO: 3
(IGF2-A1-7), or SEQ ID NO: 4 (IGF2 V43M), or a nucleic acid sequence at least
about 75%, or
80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence identity to any of SEQ ID
NOs: 2, 3
or 4.
87. The nucleic acid sequence of any of paragraphs 74-86, wherein the
nucleic acid sequence
encoding the GAA polypeptide is the human GAA gene or a human codon optimized
GAA
gene (coGAA) or a modified GAA nucleic acid sequence.
88. The nucleic acid sequence of any of paragraphs 74-87, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized for enhanced expression in
vivo.
89. The nucleic acid sequence of any of paragraphs 74-88, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce CpG islands.
90. The nucleic acid sequence of any of paragraphs 74-89, wherein the
nucleic acid sequence
encoding the GAA polypeptide is codon optimized to reduce the innate immune
response, or
reduce the CpG islands, or to reduce the innate immune response and reduce the
CpG islands.
91. The nucleic acid sequence of any of paragraphs 74-90, wherein the
nucleic acid sequence
encoding the GALA polypeptide encodes a GALA polypeptide comprising a H201L
modification
of SEQ ID NO: 10.
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92. The nucleic acid sequence of any of paragraphs 74-91, wherein the
nucleic acid encoding the
GAA polypeptide is selected from any of SEQ ID NO: 11 (full length hGAA), SEQ
ID NO: 55
(Dwight cDNA), SEQ ID NO: 56 (hGAA A1-66) or a nucleic acid sequence at least
about 75%,
or 80%, or 85%, or 90%, 01 95%, or 98%, or 99% sequence identity to any of SEQ
ID NOs: 11,
55 or 56.
93. The nucleic acid sequence of paragraph 74-92, wherein the nucleic acid
encoding the GAA
polypeptide is selected from any of SEQ ID NO: 74 (codon optimized 1), SEQ ID
NO: 75
(codon optimized 2), and SEQ ID NO: 76 (codon optimized 3), or a nucleic acid
sequence at
least about 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% sequence
identity to any of
SEQ ID NOs: 74, 75 or 76.
94. The nucleic acid sequence of paragraph 74-93, wherein the nucleic acid
is selected from any of:
SEQ ID NO: 57 (AAT-V43M-wtGAA (deltal-69aa)); SEQ ID NO: 58 (ratFN1-IGF2V43M-
wtGAA (delta1-69aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ
ID
NO: 60 (AAT-IGF2A2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN1rat- IGFA2-7-wtGAA
(delta 1-69)); SEQ ID NO: 62 (hEN1- IGRA2-7-wtGAA (delta 1-69)), SEQ ID NO: 79
(AAT hIGF2-V43M wtGAA_dell-69_Stuffer.V02); SEQ ID NO: 80 (FIBrat hIGF2-
V43M_wtGAA_del 1-69_Stuffer.V02); SEQ ID NO: 81 (FIBhum_hIGF2-V43M_wtGAA_de11-
69 Stuffer.V02); SEQ ID NO: 82 (AAT GILT wtGAA del 1-69
_______________________________________ Stuffer.V02); SEQ ID NO:
83 (FIBrat_GILT_wtGAA_del 1 -69_Stuffer.V02); SEQ ID NO: 84
(FIBhum GILT_wtGAA del 1-69_Stuffer.V02) or a nucleic acid sequence having at
least 80%,
85%, 90%, 95% or 98% identity to SEQ ID Nos: 57, 58, 59, 60, 61, 62, 79, 80,
81, 82, 83 or 84.
95. A method to treat a subject with a glycogen storage disease type II
(GSD II, Pompe Disease,
Acid Maltase Deficiency) or having a deficiency in alpha-glucosidase (GAA)
polypeptide,
comprising administering any of the recombinant AAV vector, or the rAAV genome
or the
nucleic acid sequence of any one of the previous paragraphs 1-95 to the
subject
96. The method of paragraph 95, wherein GAA polypeptide is secreted from
the subject's liver and
there is uptake of the secreted GAA by skeletal muscle tissue, cardiac muscle
tissue, diaphragm
muscle tissue or a combination thereof, wherein uptake of the secreted GAA
results in a
reduction in lysosomal glycogen stores in the tissue(s).
97, The method of any of paragraphs 95-96, wherein the administering to the
subject is selected
from any of intramuscular, sub-cutaneous, intraspinal, intracistemal,
intrathecal, intravenous
administration.
98. The method of any of paragraphs 95-97, wherein the recombinant AAV
vector is a chimeric
AAV vector, haploid AAV vector, a hybrid AAV vector or polyploid AAV vector.
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99. The method of any of paragraphs 95-97, wherein the recombinant AAV
vector is a rational
haploid vector, a mosaic AAV vector, a chemically modified AAV vector, or a
AAV vector
from any AAV serotypes.
100. The method of any of paragraphs 95-97, wherein the recombinant AAV vector
is a AAVXL32
vector or a AAVXL32.1 vector or a AAV8 vector, or a haploid AAV8 vector
comprising at
least one AAV8 capsid protein.
101. The method of paragraph 100, wherein the recombinant AAV vector is a AAV8
vector.
102. A method to treat a subject with a lysosomal storage disease (LSD),
comprising administering
any of: the recombinant AAV vector, or the rAAV genome or the nucleic acid
sequences of any
one of the previous paragraphs to the subject, wherein the AAV vector
expresses a polypeptide
selected from any polypeptide in Table 5B or Table 6B.
103. The method of paragraph 102, wherein the lysosomal storage disease (LSD)
is selected from
any of those listed in Table 5A or Table 6A.
104. The method of paragraph 102, wherein the recombinant AAV vector is a
chimeric AAV vector,
haploid AAV vector, a hybrid AAV vector or polyploid AAV vector.
105. The method of paragraph 102, wherein the recombinant AAV vector is a
rational haploid
vector, a mosaic AAV vector, a chemically modified AAV vector, or a AAV vector
from any
AAV serotypes.
106. The method of paragraph 102, wherein the recombinant AAV vector is a
AAVXL32 vector or a
AAVXL32.1 vector or a AAV8 vector, or a haploid AAV8 vector comprising at
least one
AAV8 capsid protein.
107. A cell comprising the nucleic acid sequence of any of paragraphs 74-94.
108. The cell of paragraph 107, wherein the cell is a human cell.
109. The cell of any of paragraphs 107-108, wherein the cell is a non-human
cell mammalian cell.
110. The cell of any of paragraphs 107-109, wherein the cell is an insect
cell.
111. A cell comprising the recombinant AAV vector of any of paragraphs 1-72.
112. A host animal comprising the recombinant AAV vector of any of paragraphs
1-72.
113. The host animal of paragraph 112, wherein the host animal is a mammal.
114. The host animal of paragraph 112 or 113, wherein the host animal is a non-
human mammal.
115. The host animal of paragraph 113, wherein the host animal is a human.
116. The pharmaceutical composition of paragraph 73, for use in the method of
any of paragraphs
95-101.
117. A host animal comprising a cell of any of paragraphs 107-111.
118. A host animal comprising the recombinant AAV vector of any of paragraphs
1-72.
119. The host animal of paragraph 118, wherein the host animal is a mammal.
120. The host animal of paragraph 118-119, wherein the host animal is a non-
human mammal.
121. The host animal of paragraph 119, wherein the host animal is a human.
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EXAMPLES
[00548] The following non-limiting examples are provided for illustrative
purposes only in order to
facilitate a more complete understanding of representative embodiments now
contemplated. These
examples are intended to be a mere subset of all possible contexts in which
the AAV virions and
rAAV vectors may be utilized. Thus, these examples should not be construed to
limit any of the
embodiments described in the present specification, including those pertaining
to AAV virions and
rAAV vectors and/or methods and uses thereof. Ultimately, the AAV virions and
vectors may be
utilized in virtually any context where gene delivery is desired.
EXAMPLE 1: Construction of the rAAV genome
1005491Numerous rAAV genomes were constructed using Gibson cloning
methodology. The
following rAAV genomes were generated: AAV_LVR412_EU (SEQ ID NO: 154),
ssAAV LVR412WT-hGAA Askl3io CHATHAM (SEQ ID NO: 155), AAV-LVR412Stuffer (SEQ
ID NO: 156), AAV_LVR422_EU (SEQ ID NO: 157), AAV-LVR422_Stuffer (SEQ ID NO:
158),
ssAAV_LVR412 WT-hGAA CHATHAM (SEQ ID NO: 159), ssAAV_LSP_WT-hGAA-
CHATHAM (SEQ ID NO: 160), SEQ ID NO: 57 (AAT-V43M-wtGAA (deltal-69aa)); SEQ ID
NO:
58 (ratFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 59 (hFN1-IGF2V43M-wtGAA
(deltal-
69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA (delta 1-69)); SEQ ID NO: 61 (FN1rat-
IGFA2-7-
wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1- IGFA2-7-wtGAA (delta 1-69)). While
some rAAV
vectors comprises a nucleic acid sequence encoding the wtGAA polypeptide, one
can readily replace
the wtGAA with a nucleic acid sequence encoding a modified GAA nucleic acid
sequence as
disclosed herein.
1005501Gibson cloning involves cloning blocks (e.g., 3 blocks) of nucleic acid
sequences together.
The general protocol is as follows: the following reagents are combined into a
single-tube reaction (i)
Gibson Assembly Master Mix (Exonuclease, DNA polymerase, DNA Ligase, buffer)
(ii) DNA inserts
(Blocks 1-3) with 15-25 bp of homologous ends (see, FIG. 6) (iii) Linearized
DNA backbone with
15-25 bp of homologous ends to the outermost DNA inserts (see, FIG. 6). The
reaction is incubated at
50oC for 15 ¨60 minutes. The reaction mix is transformed into competent cells
and plated on
Kanamycin agar plates. Minipreps of hilly-assembled plasmid DNA are screened
via restriction
digestion and /or colony PCR analysis and verified by DNA sequencing analysis.
Verified clone is
expanded for maxiprep production and transiently transfected in a rAAV
producer cell line alongside
the Adenovirus helper, XX680 Kan, and the appropriate Rep/Cap helper to
produce rAAV. FIG 6
show the cloning nucleic blocks to generate exemplary rAAV genomes.
[00551] While FIGS 7-9 show wtGAA(A1-69) is an exemplary GAA enzyme, this
nucleic acid
sequence can easily be replaced by one of ordinary skill with a nucleic acid
sequence encoding GAA
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that has been codon optimized for enhanced expression in vivo, and/or to
reduce immune response,
and/or to reduce CpG islands, and/or has a H201L modification. Accordingly,
while Fig. 9A-9E show
exemplary constructs with wild type GAA (wtGAA), one can readily replace the
nucleic acid
encoding the wtGAA with a nucleic acid sequence encoding a modified GAA
nucleic acid sequence
as disclosed herein, e.g., SEQ ID NO: 132. Also shown in the cloning blocks
exemplified in FIGS 7-3
is a generation of a rAAV genome a 3 amino acid (3aa) spacer nucleic acid
sequence located 3' of the
nucleic acid sequence encoding the IGF2(V43M) or IGF2A2-7 targeting peptide
and 5'of the nucleic
acid encoding a GAA enzyme, and a stuffer nucleic acid sequence a stutter
sequence (referred to in
FIGS. 7-8 as a "spacer" sequence) which is located 3' of the polyA sequence
and 5' of the 31TR
sequence.
EXAMPLE 2: generating rAAV vectors
1005521The rAAV genomes were packed into capsids to generate rAAV vectors
using a rAAV
producing cell line. Solely for proof of principal of rAAV vector
construction, the capsids used were
AAV3b capsids.
1005531Making rAAV in the rAAV producing cell line: triple transfection
technique was used to
make rAAV in a suspension rAAV producer cell line, which can be scaled up for
making clinical
grade vector. Alternatively, different plasmids can be used, e.g., 1) pXX680 -
ad helper and 2) pXR3
the Rep and Cap 3) and the Transgene plasmid (ITR¨transgene-ITR).
1005541The rAAV genomes generated in Example 1 are used to generate rAVV
vectors using a
rAAV producing cell line, according to the methods as described in US patent
9,441,206, which is
incorporated herein in its entirety by reference. In particular, rAAV vectors
or rAAV virions are
produced using a method comprising: (a) providing a rAAV producing cell line
an AAV expression
system; (b) culturing the cells under conditions in which AAV particles are
produced; and (c)
optionally isolating the AAV particles. Ratios of triple transfection of the
plasmid and transfection
cocktail volumes can be optimized, with varying plasmid ratios of XX680, AAV
rep/cap helper and
TR plasmid to determine the optimal plasmid ratio for rAAV vector production.
1005551ln some instances, the cells are cultured in suspension under
conditions in which AAV
particles are produced. In another embodiment, the cells are cultured in
animal component-free
conditions. The animal component-free medium can be any animal component-free
medium (e.g.,
serum-free medium) compatible with the rAAV producer cell line. Examples
include, without
limitation, SFM4Transfx-293 (Hyclone), Ex-Cell 293 (JRH Biosciences), LC-SFM
(Invitrogen), and
Pro293-S (Lonza). Conditions sufficient for the replication and packaging of
the AAV particles can
be, e.g., the presence of AAV sequences sufficient for replication of an rAAV
genome described
herein and encapsidation into AAV capsids (e.g., AAV rep sequences and AAV cap
sequences) and
helper sequences from adenovirus and/or heipesvirus.
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1005561Bacterial DNA sequences from the plasmid backbone can be packaged into
AAV capsids
during manufacturing of the recombinant AAV vectors leading to activations of
the innate immune
system through its interaction with TLR9 (Akira, 2006; Chadeuf, 2005; Wright,
2014). Various
technologies can be used to eliminate plasmid backbone sequences in
recombinant AAV preparations,
for example minieircles which have limited scalability (Schnodt, 2016).
Another method to avoid
bacterial DNA sequence in the plasmid backbone is to use closed ended linear
duplex DNA, which
includes a range of DNA replication technology, including but not limited to
doggy bone DNA
(dbDNATm) for specifically manufacturing of recombinant AAV vectors. Using
closed ended linear
duplex DNA, such as dbDNATM eliminates the bacterial backbone and has been
used to produce
vaccines and lentivirus (Walters et al, 2014; Scott et al, 2015; ICarda et al,
2019) and was shown to be
unable to trigger TLR9 responses by DNA vaccine developers.
1005571Accordingly, in alternative embodiments, generation of rAAV vectors for
use in the methods
and compositions as disclosed herein can be performed using closed ended
linear duplex DNA,
including but not limited to Doggybone technology (dbDNATm), as disclosed in
US Application
2018/0037943 and ICarbowniczek et al., Bioinsights, 2017, which is
incorporated herein in its entirety
by reference. In brief, a plasmid for AAV production using a closed ended
linear duplex DNA
technology can comprise the ITRs, promoter and gene of interest, e.g., GAA as
disclosed herein, is
flanked by a 56bp palindromic protelomerase recognition sequence. The plasmid
is denatured, and in
the presence of a Phi29 DNA polymerase, and appropriate primers, Phi29
initiates rolling circle
amplification (RCA), creating a double stranded cancatameric repeats of the
original construct. When
protelomerase is added, binding of the palindromic protelomerase recognition
sequences occurs and
cleavage-joining reaction occurs to result in a monomeric double stranded (ds)
linear covalently
closed DNA construct. Addition of common restriction enzymes remove the
undesired DNA plasmid
backbone sequence and digestion with exonuclease activity, resulting in dbDNA
which can be size
fractionated to isolate the dbDNA sequence encoding the ITRs, promoter and
gene of interest. An
exemplary plasmid for generation of rAAV vectors using closed ended linear
duplex DNA such as
dbDNATM technology, comprises in the following 5' to 3' direction: 5'-
protelomerase RS, 51TR,
LSP promoter, hGAA, 3'UTR, hGH poly(A), 3' ITR, 3'-protelomerase RS (sense
strand), where the
sense strand is linked to the complementary antisense strand for a stranded
(ds) linear covalently
closed DNA construct. The use of closed ended linear duplex DNA, e.g., doggy
bone DNA
(dbDNATM) as a starting material for the manufacturing of an AAV vector for
use in the methods and
composition as disclosed herein eliminates the bacterial backbone used to
propagate the plasmid
containing AAV vector with an inability for the product to trigger Toll-like
receptor 9 (TLR9)
responses. Use of closed ended linear duplex DNA technology for manufacturing
further reduce the
risk for liver enzyme elevations observed at a dose of 1.6E13 vg/kg in
patients with Pompe disease.
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EXAMPLE 3: Assessing rAAV vectors
1005581 Whole Blood Clearance. FIG. 1 shows the results derived from an
experiment where 3X1012
vg/kg of different AAV serotypes (AAV3b, AAV3ST, AAV8, AAV9) were injected
intravenously
into 3 kg seronegative male macaques. The macaques were euthananized 60 days
post administration
of the different AAV serotypes. Vector genomes were searched in whole blood
and results indicated
that AAV3b was cleared within a week and was undetectable at sacrifice,
whereas AAV8 and AAV9
were still detectable in whole blood when the macaques were sacrificed.
1005591Liver Specific Vector Potency: FIG. 2 shows the results derived from an
experiment where
3X1012 vg/kg of different AAV serotypes (AAV3b, AAV3ST, AAV8, AAV9) were
injected
intravenously into 3 kg seronegative male macaques. The macaques were
euthananized 60 days post
administration of the different AAV serotypes. Vector genomes were quantified
in each of the three
lobes of the liver from each of the macaques. The limit of quantitation was
0.002 vg/dg. Based on
the results presented in Figure 2, AAV3b was found to be a potent liver
vector. AAV3b is more liver
specific than AAV8 and cleared from the blood more rapidly than AAV9. The
AAV3ST mutant did
not provide any significant beneficial affect.
EXAMPLE 4: measuring secretion of GAA into the supernatant and GAA uptake
assays
1005601 Measuring GAA in supernatant
1005611 Accordingly, the rAAV genomes generated in Example 1 are tested for
secretion of GAA
polypeptide into the supernatant. Measurement of GAA in the supernatant can be
assessed using a 4-
methyl-umbelliferyl-alpha-D-glucoside (4-MU) substrate (4-MU assay), as
described in Kikuchi et al.
(Kikuchi, Tateki, et al. "Clinical and metabolic correction of Pompe disease
by enzyme therapy in
acid maltase-deficient quail." The Journal of clinical investigation 101.4
(1998): 827-833.).
1005621 In brief, a rAAV producer cell line can be transfected with rAAV
genomes
AAV LVR412_EU (SEQ ID NO: 154), ssAAV_LVR412WT-hGAA_AskBio_CHATHAM (SEQ
ID NO: 155), AAV-LVR412Stuffer (SEQ ID NO: 156), AAV_LVR422_EU (SEQ ID NO:
157),
AAV-LVR422 Stuffer (SEQ ID NO: 158), ssAAV LVR412 WT-hGAA CHATHAM (SEQ ID NO:
159), ssAAV_LSP WT-hGAA-CHATHAM (SEQ ID NO: 160), SEQ ID NO: 57 (AAT-V43M-
wtGAA (deltal-69aa)); SEQ ID NO: 58 (ratEN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ
ID NO: 59
(hEN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA (delta
1-69));
SEQ ID NO: 61 (FN1 rat- IGFA2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1-
IGFA2-7-wtGAA
(delta 1-69)). GAA activity is measured based on the % of initial activity
(t=0) over 24 hours.
Samples were assayed for GAA enzyme activity based on the hydrolysis of the
fluorogenic substrate
4-MU-a-glucose at 0, 3, 6 and 24 hours. The GAA activity was expressed as % of
initial activity, i.e.
residual activity.
1005631 Alternatively, after harvest, culture supernatants were partially
purified by WC
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chromatography. All samples were treated with PNGase prior to electrophoresis.
The expression of
GAA polypeptides by the cells can be assessed using SDS-PAGE and
immunoblotting.
[00564] GAA uptake Assays & measuring uptake of GAA in tissues.
1005651 Next, the rAAV genomes generated in Examples 1 and 2 are tested for
retention of uptake
activity into cells. For example, a rAAV producer cell line can be transfected
with rAAV genomes
AAV LVR412 EU (SEQ ID NO: 154), ssAAV LVR412WT-hGAA AskBio CHATHAM (SEQ
ID NO: 155), AAV-LVR412Stuffer (SEQ ID NO: 156), AAV LVR422 EU (SEQ ID NO:
157),
AAV-LVR422 Stuffer (SEQ ID NO: 158), ssAAV LVR412 WT-hGAA CHATHAM (SEQ ID NO:
159), ssAAV_LSP WT-hGAA-CHATHAM (SEQ ID NO: 160), SEQ ID NO: 57 (AAT-V43M-
wtGAA (deltal-69aa)); SEQ ID NO: 58 (ratFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ
ID NO: 59
(hFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA (delta
1-69));
SEQ ID NO: 61 (FN1rat- IGFA2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1- IGFA2-
7-wtGAA
(delta 1-69)).
[00566] A 4-MU assay (as described above) can be to assess uptake of rhGAA
into mammalian
cells is described in US patent Application U52009/01 17091A1, which is
incorporated herein in its
entirety by reference. rAAV vectors or rAAV genomes generated in Examples 1
and 2 are incubated
in 20 ill reaction mixtures containing 123 mM sodium acetate pH 4.0 with 10 mM
4-
methylumbelliferyl a-D-glucosidase substrate (Sigma, catalog #M-9766).
Reactions were incubated at
37 C. for 1 hour and stopped with 200 Id of buffer containing 267 mM sodium
carbonate, 427 mM
glycine, pH 10.7. Fluorescence was measured with 355 nm excitation and 460 nm
filters in 96-well
microtiter plates and compared to standard curves derived from 4-
methylwnbelliferone (Sigma,
catalog #M1381). 1 GAA 4 MU unit is defmed as 1 nmole 4-methyltunbelliferone
hydrolyzed/hour.
Specific activities of exemplary rAAV genomes in fibroblast cells are
assessed, e.g.,
AAV LVR412_EU (SEQ ID NO: 154), ssAAV_LVR412WT-hGAA_AskBio_CHATHAM (SEQ
ID NO: 155), AAV-LVR412Stuffer (SEQ ID NO: 156), AAV_LVR422_EU (SEQ ID NO:
157),
AAV-LVR422 Stuffer (SEQ ID NO: 158), ssAAV LVR412 WT-hGAA CHATHAM (SEQ ID NO:
159), ssAAV LSP WT-hGAA-CHATHAM (SEQ ID NO: 160), SEQ ID NO: 57 (AAT-V43M-
wtGAA (deltal-69aa)); SEQ ID NO: 58 (ratFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ
ID NO: 59
(hFN1-IGF2V43M-wtGAA (deltal-69aa)); SEQ ID NO: 60 (AAT-IGF2A2-7-wtGAA (delta
1-69));
SEQ ID NO: 61 (FN1rat- IGFA2-7-wtGAA (delta 1-69)); SEQ ID NO: 62 (hFN1- IGFA2-
7-wtGAA
(delta 1-69)). The enzymatic activity of IGF2-GAA fusion polypeptides andior
SS-IGF2-GAA double
fusion polypeptide are assessed and compared to a GAA (wtGAA) polypeptide by
itself (i.e., without
a heterologous signal peptide or IGF2 targetting peptide).
[00567] Cell-based uptake assays can also be performed to demonstrate the
ability of IGF2-tagged
or untagged GAA to enter the target cell. Rat L6 myoblasts are plated at a
density of I x105 cells per
well in 24-well plates 24 hours prior to uptake. At the start of the
experiment, media is removed from
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the cells and replaced with 0.5 ml of uptake media which contains the rAAV
vectors generated in
Examples 1 and 2. In order to demonstrate specificity of uptake, some wells
additionally contained the
competitors M6P (5mM final concentration) and/or IGF2 (18 pg/tn1 final
concentration), After 18
hours, media is aspirated off of cells, and cells are washed 4 times with PBS.
Then, cells are lysed
with 200 nl CelLytic MTM lysis buffer. The lysate is assayed for GAA activity
as described above
using the 4 MU substrate. Protein is determined using the Pierce BCATM Protein
Assay Kit.
[00568] A typical uptake experiment is performed in CHO cells, although other
cell lines and
myoblast cell lines can be used. It is expected that uptake of the GAA
polypeptides into Rat L6
myoblasts will be virtually unaffected by the addition of a large molar excess
of M6P, whereas uptake
is expected to be significantly abolished by excess IGF2. In contrast, it is
expected that uptake of
wtGAA to be significantly abolished by addition of excess M6P but virtually
unaffected by
competition with IGF2. In addition, it is expected that uptake of IGF2V43M-
wtGAA and IGFdelta2-
7wtGAA will be unaffected significantly by excess IGF2.
EXAMPLE 5 Half-Life of GAA in Rat L6 Myoblasts
[00569] An uptake experiment was performed as described above (see Example 3 &
4) with the
rAAV vectors produced in Example 1 and 2 in rat L6 myoblasts. After 18 hours,
media from cells
transfected with the rAAV vectors was aspirated off and the cells were washed
4 times with PBS. At
this time, duplicate wells were lysed (Time 0) and lysates were frozen at ¨80.
Each day thereafter,
duplicate wells were lysed and stored for analysis. After 14 days, all of the
lysates were assayed for
GAA activity, to assess the half-lives, and assess if, once inside cells, the
IGF2-tagged GAA enzyme
persists with similar kinetics to untagged GAA.
EXAMPLE 6: Processing of GAA after Uptake
[00570] Mammalian GAA typically undergoes sequential proteolytic processing in
the lysosome as
described by Moreland et al. (2005) J. Biol. Chem., 280:6780-6791 and
references contained therein.
The processed protein gives rise to a pattern of peptides of 70 kDa, 20 kDa,
10 kDa and some smaller
peptides. To determine whether IGF2-GAA fusion polypeptide and/or SS-IGF2-GAA
double fusion
polypeptide is processed similarly to the untagged GAA, aliquots of lysates
from the above uptake
experiment were analyzed by Western blot using a monoclonal antibody that
recognizes the 70 kDa
IGF2 peptide and larger intermediates with the IGF2 tag. A similar profile of
polypeptides identified
in this experiment indicates that once entering the cell, the IGF2 targeting
peptide is lost and the
IGF2-GAA polypeptide is processed similarly to untagged GAA, which
demonstrates that the IGF2
targeting peptide has little or no impact on the behavior of GAA once it is
inside the cell.
EXAMPLE 7: Pharmacokinetics
[00571] Pharmacokineties of IGF2-GAA fusion polypeptide and/or SS-IGF2-GAA
double fusion
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polypeptide produced by the rAAV vectors can be measured in wild-type 129
mice. 129 mice are
injected with the rAAV vectors generated in Example 1 and 2. Sertun samples
are taken preinjection
and at 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 4 hours, and 8 hours
post injection. The
animals are then sacrificed. Serum samples are assayed by quantitative western
blot. The half-lives for
the GAA from rAAV vectors expressing IGF2-GAA fusion polypeptide or SS-IGF2-
GAA double
fusion polypeptide are assessed to determine if the IGF2 fused GAA polypeptide
is cleared from the
circulation excessively rapidly.
EXAMPLE 8: Tissue Half-Life of GAA
[00572] The objective of this experiment was to deterniine the rate at which
GAA ctivity is lost once
the IGF2-GAA fusion polypeptide or SS-IGF2-GAA double fusion polypeptide
expressed from the
rAAV vector reaches its target tissue. In the Pompe mouse model, MYOZYMEX
appears to have a
tissue half-life of about 6-7 days in various muscle tissues (Application
Number 125141/0 to the
Center for Drug Evaluation and Research and Center for Biologics Evaluation
and Research,
Phaimacology Reviews).
[00573] Pompe mice (Pompe mouse model 6neo/6neo as described in Raben (1998)
JBC,
273:19086-19092, the disclosure of which is hereby incorporated by reference)
are injected in the
jugular vein with the rAAV vectors generated in Examples 1 and 2. Mice are
then sacrificed at 1, 5,
10, and 15 days post injection. Tissue samples were homogenized and GAA
activity measured
according to standard procedures. The tissue half-life of GAA activity from
IGF2-GAA fusion
polypeptide and/or SS-IGF2-GAA double fusion polypeptide and the untagged GAA
are calculated
from the decay curves in different tissues (e.g., quadriceps tissue; heart
tissue; diaphragm tissue; and
liver tissue), and the half-life in each tissue calculated. This can be
compared to the half-life in rat L6
myoblasts to determine if, once inside cells in Pompe mice, IGF2-GAA fusion
polypeptide and/or SS-
IGF2-GAA double fusion polypeptide expressed from the rAAV vectors described
herein appears to
persist with kinetics similar to the untagged GAA. Furthermore, the knowledge
of the decay kinetics
of the IGF2-GAA fusion polypeptide and/or SS-IGF2-GAA double fusion
polypeptide can help in the
design of appropriate dosing intervals.
EXAMPLE 9: Uptake of IGF2-GAA fusion polypeptide ancUor SS-IGF2-GAA double
fusion
polypeptide into Lysosomes of C2C12 Mouse Myoblasts
[00574] C2C12 mouse myoblasts grown on poly-lysine coated slides (BD
Biosciences) are
transdue,ed with the rAAV vectors produced in Examples 1 and 2. After washing
the cells, the cells
are then incubated in growth media for 1 hour, then washed four times with D-
PBS before fixing with
methanol at mom temperature for 15 minutes. The following incubations were all
at room
temperature, each separated by three washes in D-PBSõ Slides are
permeabilizecl with 0.1% triton X-
100 for 15 minutes, then blocked with blocking buffer (10% heat-inactivated
horse senun (Invitrogen)
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in D-PBS). Slides are incubated with primary mouse monoclonal anti-GAA
antibody 3A6-1F2
(1:5,000 in blocking buffer), then with secondary rabbit anti-mouse IgG AF594
conjugated antibody
(Invitrogen A11032, 1:200 in blocking buffer). A FITC-conjugated rat anti-
mouse LAMP-1 (BD
Pharmingen 553793, 1:50 in blocking buffer) is the incubated. Slides are
mounted with DAPI-
containing mounting solution (Invitrogen) and viewed with a Nikon Eclipse 80i
microscope equipped
with fluorescein isothiocyanate, texas red and DAPI filters (Chroma
Technology). Images can be
captured with a photometric Cascade camera controlled by MetaMorph software
(Universal Imaging),
and merged using Photoshop software (Adobe). Co-localization of signal
detected by anti-GAA
antibody with signal detected by antibody directed against a lysosomal marker,
LAMP1 can be
assessed to demonstrates that IGF2-tagged GAA is delivered to lysosomes.
EXAMPLE 10: Assessing the treatment of the rAAV vectors in a Pompe mouse model
and reversing
Pompe pathology
[00575] The rAAV vectors generated in Example 1 can be assessed in Pompe mouse
mode, e.g.,
according to the methods described in Peng et al.,. "Reveglucosidase alfa (BMN
701), an IGF2-
Tagged rhAcid a-Glucosidase, Improves Respiratory Functional Parameters in a
Murine Model of
Pompe Disease." Journal of Pharmacology and Experimental Therapeutics 360.2
(2017): 313-323),
which is incorporated herein in its entirety by reference.
[00576] Any Pompe mouse model can be used to assess the effect of the rAAV
vectors at treating
Pomoe disease. One mouse model of Pompe is described in Raben etal., JBC,
1998; 273(30); 19086-
19092, which describes a disrupted GAA mouse model, and recapitulates critical
features of both the
infantile and the adult forms of the disease. In other instances, a Pompe
mouse model (Sidman et at.,
2008) can be used, as well as a strain of mice with a disrupted acid a-
glucosidase gene (B6;129-
GAAtmlRabn/J; Pompe) (Jackson Laboratory, Bar Harbor, ME). The Pompe mice
develop the same
cellular and clinical characteristics as in human adult Pompe disease (Raben
et al., 1998). Animals are
maintained in a 12-hour light/dark cycle, provided with fresh water and
standard rodent chow ad
libitum.
[00577] 4.5-5 month old Pompe mice can be administered the rAAV vectors
described herein, and
evaluated for glycogen clearance after administration for 4 or more weeks.
Following a macroscopic
assessment, the heart (left ventricle), quadriceps, diaphragm, psoas, and
soleus muscles were
collected, weighed, snap-frozen in liquid nitrogen, and stored at -60 to -90 C
prior to a quantitative
analysis of glycogen-derived glucose. Muscles were homogenized in buffer (02 M
Na0Ac/0.5%
NP40) on ice using ceramic spheres. Amyloglucosidase was added to clarified
lysates at 37 C to
digest glycogen into glucose for subsequent colorimetric detection (430 rmi,
SpectraMax; Molecular
Devices, Sunnyvale, CA) using a peroxidase-glucose oxidase enzyme reaction
system (Sigma-
Aldrich, St. Louis, MO). Paired samples are also measured without
amyloglucosidase to correct for
endogenous tissue glucose that was not in glycogen form at harvest. Glucose
values were extrapolated
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from a six-point standard curve. The measured glucose concentration (mg/ml) is
proportional to the
glycogen concentration of the sample and is converted to mg glycogen/g tissue
by adjusting for the
homogenization step (5 pi buffer added per gram of tissue).
[00578] The effect of rAAV vectors described herein on individual mouse muscle
glycogen levels
can be evaluated using Phoenix-WinNonlin classic PD modeling (Phoenix build
version 6.4; Certara,
L.P., Princeton, NJ). Results can be obtained for hGAA in heart, diaphragm,
quadriceps, psoas, and
soleus muscles. For pharmacokinefic analysis, WT mice can be administered the
rAAV vectors
generated in Example 1 and blood samples collected as terminal cardiac
punctures at predose, 0.083,
0.5, 1, 2, and 4 hours postdose. Plasma hGAA concentrations can be quantified
using a bridging
electrochemiluminescent method with an LOQ of 100 ng/ml. Briefly, 0.5 tighnl
ruthenium-labeled
anti-rhGAA (affinity purified goat polyclonal) and 0.5 pg/m1 biotin-labeled
anti-IGF2 (MAB792;
R&D Systems, Minneapolis, MN) can be combined with ICEDTA plasma samples
diluted 1:10 in
buffer [Starting Block T20 (PBS); ThermoFisher Scientific, Sunnyvale, CA] and
incubated for 1 hour
before transfer to a blocked streptavidin assay plate (Meso Scale Diagnostics,
Rockville, MD). After a
30-minute incubation, the plate is washed, lx Read Buffer T (Meso Scale
Diagnostics) was added,
and the electrochemiluminescent signal read on an SECTOR Imager 2400 (Meso
Scale Diagnostics).
hGAA concentrations can be extrapolated from a standard curve.
[00579] Alternatively, Heart and Diaphragm tissue homogenates can be harvested
and rhGAA
activity measured using the fluorogenic substrate (4-MUG).
[00580] The therapeutic effect of the GAA polypeptide produced using rAAV
vectors generated in
Examples 1 and 2 herein can be compared wt GAA in vivo. A study can be
performed to compare the
ability of a rAAV vector disclosed in Example 1 to that expressing a non-
tagged wt GAA to clear
glycogen from skeletal muscle tissue in Pompe mice (e.g., Pompe mouse model
6neo/6neo animals
were used (Raben (1998) JBC 273:19086-19092)). Groups of Pompe mice (5/group)
received IV
injections of one of two doses of wt GAA or a rAAV vector generated in Example
1 or vehicle. Five
untreated animals can be used as control, and receive four weekly injection of
saline solution.
Animals receive oral diphenhydromine, 5 mg/kg one hour prior to injections 2,
3, and 4. Mice were
sacrificed one week after the injection, and tissues (diaphragm, heart, lung,
liver, soleus, quadriceps,
gastrocnemius, TA, EDL, tongue) are harvested for histological and biochemical
analysis. Glycogen
content in the tissue homogenates can be measured using A. niger
amyloglucosidase and the Amplex
Red Glucose assay kit, and GAA enzyme levels assessed in different tissue
homogenates using using
standard procedures.
[00581] Glycogen content in tissue homogenates can be measured using A. niger
arnyloglucosidase
and the Amplex Red Glucose assay kit (Invitrogen) essentially as described by
Zhu et al. (2005)
Biochem J., 389:619-628.
[00582] It is expected that the rAAV vector ss-IGF2-GAA rAAV as described
herein and produced
by the methods of Examples 1 and 2 will have more secretion followed by uptake
into muscle and
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greater therapeutic effect in the Pompe mouse model as compared to a IGF2-GAA
rAAV (i.e.,
without the secretory signal sequence), which is expected to be greater than
wtGAA rAAV vector
(i.e., without either of the heterologous secretory signal and the IGF2
targeting peptide), and/or
MYOZYMEO. It is further expected that the rAAV vector comprising modified GAA
as disclosed
herein e.g., comprising GAA with H201L mutation is therapeutically more
effective in the Pompe
mouse model than the rAAV comprising unmodified GAA e.g., wtGAA. Given the
established
Pompe model, these results are expected to translate into the clinic and
correlate with therapeutic
effect for the treatment of Pompe disease.
EXAMPLE 11: Clearance of Glycogen In Vivo
[00583] The objective of this experiment is to determine the rate at which
glycogen is cleared from
heart tissue of Pompe mice after a single injection of rAAV vector expressing
IGF2-GAA fusion
polypeptide and/or SS-IGF2-GAA double fusion polypeptide produced in Examples
1 and 2.
[00584] Pompe mice (Pompe mouse model 6neo/6neo as described in Raben (1998)
JBC,
273:19086-19092, the disclosure of which is hereby incorporated by reference)
are injected in the
jugular vein with a rAAV vector expressing produced in Examples 1 and 2. Mice
were sacrificed at
1, 5, 10, and 15 days post injection. Heart tissue samples are homogenized
according to standard
procedures and analyzed for glycogen content. Glycogen content in these tissue
homogenates is
measured using A. niger amyloglucosidase and the Amplex Red Glucose assay kit
(Invitrogen)
essentially as described by Zhu et al. (2005) Biochem J., 389:619-628.
Assessment of the heart tissue
from mice can determine if there is almost complete clearance of glycogen in
the mice administered
rAAV vector expressing IGF2-GAA fusion polypeptide and/or SS-IGF2-GAA double
fusion
polypeptide produced in Examples 1 and 2 as compared to mice administered a
rAAV where GAA
was not fused to a IGF2 targeting peptide and/or SS as described herein, where
only a small change in
glycogen content would indicate minimal clearance.
EXAMPLE 12: Exemplary liver specific promoters
[00585] Exemplary liver-specific promoters selected from Table 4 herein, or
selected from those
disclosed in Tables 4 herein, were cloned upstream of the luciferase reporter
gene followed by SV40
late PolyA signal into a vector with a backbone having properties essentially
identical to pUC19. In
particular, experiments assessing promoters SP0412 and SP0422 were cloned into
constructs for
generation of rAAV. DNA preparations of the plasmids were transfected into
either Huh7 (a hepato-
cellular carcinoma cell line), HeLa (an immortal cell line derived from
cervical cancer) or HEIC293
(human embryonic kidney cells) to asess transcriptional activity. Huh-7 cells
were sourced from
JCRB Cell Bank (JCRB0403), HeLa and HEIC293 were sourced from ECACC cell bank.
All cell
lines were grown and maintained according to the cell banks' recommendations.
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1005861Transfections were performed in 48 well plates in triplicate using
FuGene HD Transfection
Reagent (Promega 14E2311) at a DNA:FuGene HD ratio of 1:1.1. Luciferase
activity was measured
24 hours after transfection. Cells were washed with phosphate buffered saline
(PBS), lysed in 100 JAI
Passive Lysis Buffer (Promega #E194A) and stored at -80 'DC overnight.
Luciferase activity was
quantified using the Luciferase Reporter 1000 assay system (Promega #E4550)
following
manufacturer's guidelines in 10 1.11 of lysate using 96 well flat bottom solid
white Microplate
FluoroNunc plates (ThermoFisher #236105) and luminescence quantified in a
FLUOstar Omega plate
reader (BMG Labtech) machine.
1005871The above luciferase methods are conventional in the art, and similar
techniques have been
described extensively in the literature, e.g. in Alam and Cook, "Reporter
Genes: Application to the
Study of Mammalian Gene Transcription", ANALYTICAL BIOCHEMISTRY 188,245-254
(1990).
1005881Data
1005891The sequences of exemplary liver-specific promoters used are shown in
Tables 4 herein. The
ability of these synthetic liver-specific promoters to drive expression in
liver cells was benchmarked
against the ubiquitous CMV_IE and CBA promoters, and also against the known
liver specific
promoter LPL All of the synthetic promoters according to the invention showed
higher activity than
the LP1 promoter in Huh7 cells (data not shown). When these promoters were
counter-screened in
non-liver-derived HEK293 and HeLa cells, they showed negligible activity
compared to the
ubiquitously active promoters CMV_IE and CBA (data not shown).
1005901The activity of the liver-specific promoters (i.e. all of the promoters
set out in Tables 4) were
also tested in Huh7 cells using the materials and methods essentially as
described in above. However,
in this case the activity of the liver-specific promoters was compared to the
activity of the promoter
'TBG (SEQ ID NO; 435), as TBG was found to have higher and more consistent in
vitro expression
than LP1. It should be noted that 'TBG is an extremely powerful liver-specific
promoter, and thus a
promoter which shows expression which is less than TBG may still be extremely
useful. In particular,
liver-specific promoters disclosed in Table 4, or functional variants thereof,
which are shorter than
TBG, but which still demonstrate high levels of activity (e.g. 15%, more
preferably 25%, 50%, or
75% of the activity of TBG of SEQ ID NO: 435 or higher) are of particular
interest.
1005911The specificity of the liver-specific promoters for liver cells was
also tested using non-liver
HEK293 cells, using the materials and methods described in Example 2. The
activity of the liver-
specific promoters is expressed compared to the activity of CMV-IE (SEQ ID NO:
433) (TBG and
LP1 are liver-specific and thus not particularly active in HEK293 cells).
'Relative activity' in the
graphs showing the specificity of the liver-specific promoters tested in
HEK293 cells is the activity of
the named promoter expressed as a ratio to the activity of CMV-IE, wherein 1
is the same activity as
the CMV-IE promoter (SEQ ID NO: 433), more than 1 is higher activity compared
to CMV-IE and
lower than 1 is lower activity compared to the CMV-IE promoter of SEQ ID NO:
433.
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EXAMPLE 13: Expression from exemplary liver specific promoters in vivo
1005921Certain liver-specific promoters were selected as exemplary liver-
specific promoters for
assessment in vivo. In particular, minimal promoter CRM_5P0239 (SEQ ID NO; 87)
and CRM-
SP0412 (SEQ ID NO: 86) and synthetic liver-specific promoters 5P0239 (SEQ ID
NO: 93), SP0412
(SEQ ID NO: 91) and 5P0422 (SEQ ID NO: 92) were assessed in vivo (see FIG. 10
and FIG. 13A-
13B).
1005931AAV production:
The activity of a subset of the promoters according to this invention were
tested in vivo. The synthetic
promoters included in this study were SP0239, SP0244, SP0412 and the positive
control LP1. The
reporter gene used was fLUC-T2A-EGFP, i.e. fl,UC (firefly Luciferase) fused to
mEGFP (mutant
green fluorescent protein) via T2A signal (two-way self-cleaving peptides).
pAAV_SYNP_Luc-T2A-
GFP destination vector is derived from pAAV ZsGreen1 (purchased from Clontech)
in which the
ZsGreen1 reporter was replaced by the Luc-T2A-GFP dual reporter. All DNA
plasmids were prepared
using QIAGEN Plasmid Mega Kit (Qiagen#12181, Germany) according to
manufacturer instructions.
1005941A rAAV producer cell line was cultured in Culture dish, Tissue culture
treated, 145mm
(Greiner Bio-One Ltd # 639160, UK) in Dulbecco's modified Eagle's medium, high
glucose,
GlutaMAX supplement (Gibco (Life Technologies) ft 61965-059, UK) supplemented
with 10% (v/v)
fetal bovine serum (Sigma# F7524,UK), and incubated at 37 C and 5% CO2. Other
reagents for cell
culture were purchased from Invitrogen-UK and plasticware form Life
Technologies.
1005951All AAV vectors that were used in this study were pseudotyped in AAV9
capsid. A rAAV
producer cell line was co-transfected with plasmids wherein the reporter gene
was controlled by
different promoters alongside a plasmid encoding the helper functions to allow
virus propagation
(pDG9). The rAAV producer cell line were transfected using Polyethylenimine
(PEI) (Sigma-
Aldrich# 764604, UK) at stock concentration of( lug/ul) using molar ratio of
1:3 (DNA:PEI).
[005961/1AVpurification and titration:
After 72 hr of ftansfection, cells were lysed and crude lysate was filtered
then purified by F1PLC
columns containing POROSTM CaptureSelectTM AAV Resin (Thermo Scientific,
#A36739) and using
AKTAprime plus (High performance liquid chromatography-HPLC system (GE
Healthcare,
#11001313).
1005971The number of vector genomes was determined by qPCR titration to target
LUC cassettes
with forward primer (ACGCTGGGCTACTTGATC - SEQ ID NO: 445), reverse primer
(CGAGGAGGAGCTATTCTTG - SEQ ID NO: 446) and probe (IT TCGGGTCGTGCTCATG - SEQ
ID NO: 447) following manufacturer instructions of Luna Universal qPCR Master
Mix (NEB#
M3003, UK) in QuantStudioTM 3 System Real-Time PCR (Thermo Fisher Scientific,
UK) and data
analysed by QuantStudio design and analysis software V1.4.1.
1005981 Animal procedures:
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[00599] Outbred 6 weeks old CD1 male mice were purchased from Charles River-
UK. They were
kept in quarantine for one week and then moved to their closed ventilation
cages and maintained in
minimal-disease facilities. They were caged at 5 mice/cage and normalized into
their weights with
food and water ad libitum. Newly housed mice were given another week for
acclimatization before
carrying out any experiments. This study was conducted under statutory Home
Office
recommendation; regulatory, ethics, and licensing procedures; and the Animals
(Scientific
Procedures) Act 1986 and following the institutional guidelines at University
College London.
1006001Animal injections:
[00601] AAV was administered to 8-week-old young adult male CD1 mice
anaesthetised with 2%-
4% isoflurane supplied in medical air (21% oxygen) (Abbotts Laboratories, UK)
in warm chamber
(Thermo Fisher Scientific, UK). The mice were injected intravenously into
lateral tail vein using an
Insulin syringe: 27 G 1/2 in., 1.0 ml (Fisher Scientific, UK). Each mouse is
injected with AAV
vectors dose of 8E+10 AAV viral genome per mouse in a final volume of 200 p.1
of physiological
saline solution. The mice were then allowed to return to normal temperature
before placing them back
into their cages.
[00602] Bioluminescence imaging
[00603] Mice were subjected to weekly whole-body bioluminescence imaging.
Where appropriate,
mice were anaesthetised with 2 4-4% isoflurane supplied in medical air (21%
oxygen) and received
an intmperitoneal injection of 300 pl of 15 mg/mL of D-luciferin potassium
salt (Syd Labs #
MB000102, US) using an Insulin syringe (Fisher Scientific, UK). D-luciferin
stock was prepared in
physiological saline (Gibco 1414190-094, UK). Mice were imaged after 5 minutes
using a cooled
charged-coupled device camera, (IVIS Lumina II machine, Perkin Elmer, UK) for
between 1 second
and 10 seconds. The regions of interest (ROI) were measured using IVIS Lumina
Living image 4.5.5
(Perkin Elmer) and expressed as photons per second per centimetre squared per
steradian
(photons/second/cm2/sr).
[00604] Data
[00605] The results of this study are shown in FIG. 10. The results are
expressed as the mean of the
luciferase bioluminescence intensity, total flux (in photons per second), for
all tested animals in each
group. Error bars are standard error of the mean. In group 'Saline' (n=10),
the animals were injected
with saline only and no luciferase bioluminescence is detected. This group is
a negative control and
indicates that no luciferase bioluminescence is detected if no luciferase
operably linked to a promoter
is injected. In group `LP1' (n=9), the animals were injected with luciferase
operably linked to the LP!
promoter (SEQ ID NO: 432) and luciferase bioluminescence is detected. This
group is a positive
control and indicates that luciferase is expressed under the control of the
LP1 promoter and can be
detected.
[00606] To test the activity of the liver-specific promoters according to this
invention, animals were
injected with a construct comprising luciferase under operably linked to two
promoters. In group
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µSP0244' (n=8), luciferase is operably linked to the SP0244 promoter. In group
'SP0239' (n=10),
luciferase is operably linked to the 5P0239 promoter (SEQ ID NO: 93).
[00607] As can be seen from FIG. 10, expression was highest from promoters
'5P0244' (SEQ ID NO:
366) and `SP0239' (SEQ ID NO: 93), as shown by higher bioluminescence
intensity than the generic
liver specific promoter `LP1' (SEQ ID NO: 432). Accordingly, promoters 5P0244
and SP0239 show
high activity in vivo and their activity is higher than the activity of LP1.
[0060811n FIGS. 13A and 13B, the expression of GAA was detected from different
rAAV vectors
comprising different LSP; LSP NEW (SEQ ID NO: 160); 412 NEW (SEQ ID NO: 159);
TYR NEW
(SEQ ID NO: 155); 412-TYR, 422 Stuffer (SEQ ID NO; 158); 422 'TTR; 412 Stuffer
(SEQ ID NO:
156) intracellularily in Huh7 cells and HEPH2 cells, demonstrating that hGAA
can be expressed from
rAAV at significant levels using SP0412 and 5P0422 promoter. Furthermore, as
shown in FIGS 13A
and 13B, the GAA expression by any of the above AAV vectors is significantly
higher than the
expression of GAA from an AAV vector comprising a generic liver specific
promoter. Importantly,
FIG. 13A and 13B show that expression of hGAA from rAAV vectors comprising
promoters 412
(SEQ ID NO: 91) and 422 (SEQ ID NO: 92) leads to high expression of hGAA in
Huh7 and HepG2
cells, as compared to the level of hGAA expression from a rAAV comprising the
LP1 promoter (SEQ
ID NO: 432) which is referred to as "LSP SS" in FIG. 13A and 13B.
[00609] This experiment demonstrates confirms that the in vitro results
obtained in Example 12 can be
achieved with significant GAA protein expression in vivo. A range of liver-
specific promoters with
varying strength can be used in the methods and compositions disclosed herein,
which can be very
useful to provide the desired level of liver-specific expression in a
therapeutic setting for the treatment
of a disease, e.g., Pompe disease.
EXAMPLE 14; Clinical study of Pompe patients
[00610] Two cohorts of 3 subjects each have been dosed with rAAV-LSP-hGAA in
clinical study
ACT-CS101, which recruited adult subjects with LOPD. Cohort 1 subjects
received 1.6E12 vg/kg and
Cohort 2 subjects received 1_6E13 vg/kg.. All subjects have been withdrawn
from enzyme replacement
therapy (ERT) (alglucosidase alfa [LUMIZYME01); Cohort 1 subjects remained on
ERT for 6
months after rAAV-LSP-hGAA administration, whereas Cohort 2 subjects were
withdrawn from ERT
at the time of rAAV-LSP-hGAA administration. The construct for the AAV8-
LSPhGAA is shown in
FIG. 3B, and is an infectious, non-replicating recombinant adeno-associated
virus (AAV) serotype 8
vecto comprising AAV2 ITRs, or a haploid AAV vector comprising capsid 8 and
another capsid
protein eg., from AAV2 (e_g., AAV2/8-LSPhGAA), expressing human acid alpha-
glucosidase
(hGAA).
[00611] Preliminary data indicate no serious adverse events as of 1 June 2020
that met the expedited
reporting rule. Reported adverse events were mostly expected/anticipated and
have been mild to
moderate in severity, transient and reversible.
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1006121Cohort 1 received 1.6E12 vg/kg which has been well tolerated by all 3
subjects, all of which
have now reached 12 months post-dose with biopsy data being available shortly.
No elevations in
aspantate transarninase (AST) or alanine transaminase (ALT) have been observed
in any subject
enrolled in Cohort 1 (1.6E12 vg/kg). All subjects received immune prophylaxis
with prednisone
starting at 60 mg/day for 4 weeks followed by a tapering regimen (5 mg/week
for 11 weeks). No
subject in Cohort 1 exhibited positive enzyme-linked immune absorbent spot
(ELISPOT) reactivity
for capsid or acid alfa-glucosidase (GAA). All subjects exhibited serum GAA
concentrations above
their respective baseline values; however, serum GAA concentrations are
decreasing over time. None
have needed to receive rescue with ERT,
1006131Cohort 2 received 1.6E13 vg/kg (dosed mid-August, mid-September and
early October 2019).
Asymptomatic elevations in AST and ALT have been observed in all subjects in
Cohort 2 beginning
on Day 46 (004), Day 41 (005), and Day 55 (006). All subjects received immune
prophylaxis with
prednisone starting at 60 mg/day; however, all subjects received augmented
doses of prednisone in
response to elevations in AST and ALT. Two subjects (004 and 005) received IV
methylprednisolone.
Two subjects in Cohort 2 had CTCAE Grade 3 laboratory elevations. Subject 004
experienced one
Grade 3 elevation in ALT (348 U/L; ULN = 63; 5x ULN =315) and one Grade 3
elevation in AST
(222 U/L; ULN = 41; 5x ULN = 205). Both Grade 3 elevations occurred on the
same day and were
considered definitely/probably related to ACTUS-101 by the Principal
Investigator. Subject 5
experienced one Grade 3 elevation in gamma-glutamyl transfemse (GOT) (311 U/L;
ULN = 50; 5x
ULN = 250) which the Principal Investigator considered possibly related to
ACTUS-101, although
more probably to prednisone. All Grade 3 elevations were transient and
asymptomatic. Subject 004
had a liver biopsy within 10 days following the Grade 3 laboratory elevations,
the results of which
indicated mild and non-specific inflammation. This subject also reported a
lipoma at Week 24, which
has been attributed to the high and long duration of steroid administration,
which has since been
tapered. All subjects exhibited positive but varying levels of ELISPOT T cell
responses to AAV8
capsid which is not always concordant in time with the noted liver enzyme
changes.
1006141Aalytical comparability studies were conducted to determine the
effects, or absence thereof,
of proposed manufacturing changes on the identity, strength, quality, purity,
or potency of the AAV8-
LSPhGAA.
1006151Analytical comparability of AAV8-LSPhGAA can be assessed using a
qualified in vitro
potency assay run at Absorption Systems Boston, LLC (ASB, Medford, MA) under
Method standard
operating procedure (SOP), which is an in vitro assay for determination of the
potency of AAV8-
LSPhGAA test samples relative to a reference standard. In brief, a 6-day assay
in 96-well HuH-7 cell
culture plate format (human hepatocyte cell line, Sekisui XenoTech, Cat. No
JCRB0403) is
performed, transduced with AAV8-LSPhGAA using a 9-point dilution scheme , and
incubated for 96
2 hours after which GAA is measured in the cell supernatant by the 4-MU
bioassay. Relative
potency is calculated with the parallel line assay model using PLA Software
3.0 (Stegmann Systems).
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1006161Dose Ranging studies:
1006171Dose-ranging studies in GAA knockout (KO) mice, which were used to
support the
pharmacodynamic effects of AAV8-LSPhGAA. Male and female GAA KO Pompe mice
(B6,129-
GaatmlRabn/J, stock no. 004154, 6neo) are be injected at 8 to 12 weeks
(modification 1 proposed 9
to 12 week prior to feedback from the agency) of age by tail vein
(intravenous) injection (bolus). A
minimum of 6 males (M) and 6 females (F) are be randomly assigned to receive
one of the test
products (AAV8-LSPhGAA) dosed at 2E11, 2E12 and 2E13 vg/kg based on Han et al
(2017 and
2019) and a control group will receive the formulation buffer only (vehicle).
Animals will be followed
for 8 weeks and then sacrificed for tissue and blood collection. Experimental
and control groups will
remain blinded to the operator(s).
1006181In life monitoring will consist of daily checks for mortality. Moribund
animals will be
removed from the study (with gross necropsy examination and tissue sampling
for histopathology
performed). Serum GAA will be measured in all groups every 2 weeks until
termination at 8 weeks
after dosing.
1006191Measurements and tissue collection at necropsy will include, Liver,
heart, quadriceps, Tibialis
A., Diaphram, Brain, Soleus, kidney, spleen, lung, and other clinical
measurements, including: (1)
Terminal body weight, (2) Organ weights for liver and heart, (3) Serum (GAA
activity) and storage at
-20 C for subsequent measurement of anti-GAA antibodies (ADA), (4) Liver,
heart, soleus,
quadricep, tibialis anterior, tongue, diaphragm, brain to be split in several
fragments. Additional
measurements includes: (4) GAA activity in serum and tissue and glycogen
content in tissues, as
measured by qualified assays (tissue will be snap frozen in liquid nitrogen
and stored at -80 C). A
target of ¨50 mg of each specified tissue will be collected for each assay,
and (5) Histopathological
examination analysis fixed in 10% neutral buffered formalin for Haemotoxylin
and Eosin (H&E)
staining on tissues as indicated above as well as any observed gross lesions
at necropsy. A subsection
of tissues will be fixed in glutaraldehyde for glycogen staining as needed
(Periodic acid-Schiff
staining - PAS), Assessment of immune responses to the transgene product by
measuring anti-GAA
antibody responses (ADA), Total DNA for vector genome copy number per diploid
genome
determination ¨ vg/ug of DNA (snap frozen in liquid nitrogen and then stored
at -80 C).
1006201 Dose-ranging studies in GAA KO mice will support in vivo comparability
between AAV8-
LSPhGAA and a modified AAV vector, modified according to the disclosure herein
(e.g., different
LSP, optimization of hGAA sequence, if no significant differences are observed
by the following
measurements: 1. Histopathology by certified veterinary pathologist in all
collected tissues (safety); 2.
GAA activity and glycogen content in heart, diaphragm and quadriceps
(efficacy) with a relative
activity of the modified AAV vector between 0.80 to 1.25 (80-125%) of AAV8-
LSPhGAA; 3. Vector
genome DNA per ug DNA in collected tissues (biodistribution) with a relative
biodistribution of a
modified AAV vector between 0.80 to 125 (80-125%) of AAV8-LSPhGAA; 4. No
change of anti-
GAA ADA.
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1006211While the present inventions have been described and illustrated in
conjunction with a
number of specific embodiments, those skilled in the art will appreciate that
variations and
modifications may be made without departing from the principles of the
inventions as herein
illustrated, as described and claimed. The present inventions may be embodied
in other specific forms
without departing from their spirit or essential characteristics. The
described embodiments are
considered in all respects to be illustrative and not restrictive.
1006221 In closing, regarding the exemplary embodiments of the present
invention as shown and
described herein, it will be appreciated that a genomic construct, comprising
an AAV (adeno-
associated virus) viral virion is disclosed and configured for delivery of AAV
vectors. Because the
principles of the invention may be practiced in a number of configurations
beyond those shown and
described, it is to be understood that the invention is not in any way limited
by the exemplary
embodiments, but is generally directed to a genomic construct, comprising an
AAV (adeno-associated
virus) viral virion apparatus and is able to take numerous forms to do so
without departing from the
spirit and scope of the invention.
1006231 Certain embodiments of the present invention are described herein,
including the best mode
known to the inventor(s) for carrying out the invention. Of course, variations
on these described
embodiments will become apparent to those of ordinary skill in the art upon
reading the foregoing
description. The inventor(s) expect skilled artisans to employ such variations
as appropriate, and the
inventor(s) intend for the present invention to be practiced otherwise than
specifically described
herein. Accordingly, this invention includes all modifications and equivalents
of the subject matter
recited in the claims appended hereto as permitted by applicable law.
Moreover, any combination of
the above-described embodiments in all possible variations thereof is
encompassed by the invention
unless otherwise indicated herein or otherwise clearly contradicted by
context.
1006241 Groupings of alternative embodiments, elements, or steps of the
present invention are not to
be construed as limitations. Each group member may be referred to and claimed
individually or in
any combination with other group members disclosed herein. It is anticipated
that one or more
members of a group may be included in, or deleted from, a group for reasons of
convenience and/or
patentability. When any such inclusion or deletion occurs, the specification
is deemed to contain the
group as modified thus fulfilling the written description of all Markush
groups used in the appended
claims.
1006251 Unless otherwise indicated, all numbers expressing a characteristic,
item, quantity,
parameter, property, term, and so forth used in the present specification and
claims are to be
understood as being modified in all instances by the term "about." As used
herein, the term "about"
means that the characteristic, item, quantity, parameter, property, or term so
qualified encompasses a
range of plus or minus ten percent above and below the value of the stated
characteristic, item,
quantity, parameter, property, or term. Accordingly, unless indicated to the
contrary, the numerical
parameters set forth in the specification and attached claims are
approximations that may vary. At the
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very least, and not as an attempt to limit the application of the doctrine of
equivalents to the scope of
the claims, each numerical indication should at least be construed in light of
the number of reported
significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical
ranges and values setting forth the broad scope of the invention are
approximations, the numerical
ranges and values set forth in the specific examples are reported as precisely
as possible. Any
numerical range or value, however, inherently contains certain errors
necessarily resulting from the
standard deviation found in their respective testing measurements. Recitation
of numerical ranges of
values herein is merely intended to serve as a shorthand method of referring
individually to each
separate numerical value falling within the range. Unless otherwise indicated
herein, each individual
value of a numerical range is incorporated into the present specification as
if it were individually
recited herein. Similarly, as used herein, unless indicated to the contrary,
the term "substantially" is a
term of degree intended to indicate an approximation of the characteristic,
item, quantity, parameter,
property, or term so qualified, encompassing a range that can be understood
and construed by those of
ordinary skill in the art.
1006261 Use of the terms "may" or "can" in reference to an embodiment or
aspect of an embodiment
also carries with it the alternative meaning of "may not" or "cannot." As
such, if the present
specification discloses that an embodiment or an aspect of an embodiment may
be or can be included
as part of the inventive subject matter, then the negative limitation or
exclusionary proviso is also
explicitly meant, meaning that an embodiment or an aspect of an embodiment may
not be or cannot
be included as part of the inventive subject matter. In a similar manner, use
of the term "optionally"
in reference to an embodiment or aspect of an embodiment means that such
embodiment or aspect of
the embodiment may be included as part of the inventive subject matter or may
not be included as part
of the inventive subject matter. Whether such a negative limitation or
exclusionary proviso applies
will be based on whether the negative limitation or exclusionary proviso is
recited in the claimed
subject matter.
1006271 When used in the claims, whether as filed or added per amendment, the
open-ended
transitional term "comprising" (along with equivalent open-ended transitional
phrases thereof such as
"including," "containing" and "having") encompasses all the expressly recited
elements, limitations,
steps and/or features alone or in combination with un-recited subject matter;
the named elements,
limitations and/or features are essential, but other unnamed elements,
limitations and/or features may
be added and still form a construct within the scope of the claim. Specific
embodiments disclosed
herein may be further limited in the claims using the closed-ended
transitional phrases "consisting of'
or "consisting essentially of' in lieu of or as an amendment for "comprising."
When used in the
claims, whether as filed or added per amendment, the closed-ended transitional
phrase "consisting of'
excludes any element, limitation, step, or feature not expressly recited in
the claims. The closed-
ended transitional phrase "consisting essentially of' limits the scope of a
claim to the expressly recited
elements, limitations, steps and/or features and any other elements,
limitations, steps and/or features
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that do not materially affect the basic and novel characteristic(s) of the
claimed subject matter. Thus,
the meaning of the open-ended transitional phrase "comprising" is being
defined as encompassing all
the specifically recited elements, limitations, steps and/or features as well
as any optional, additional
unspecified ones. The meaning of the closed-ended transitional phrase
"consisting of' is being
defined as only including those elements, limitations, steps and/or features
specifically recited in the
claim, whereas the meaning of the closed-ended transitional phrase "consisting
essentially of' is being
defined as only including those elements, limitations, steps and/or features
specifically recited in the
claim and those elements, limitations, steps and/or features that do not
materially affect the basic and
novel characteristic(s) of the claimed subject matter. Therefore, the open-
ended transitional phrase
"comprising" (along with equivalent open-ended transitional phrases thereof)
includes within its
meaning, as a limiting case, claimed subject matter specified by the closed-
ended transitional phrases
"consisting of' or "consisting essentially of" As such, embodiments described
herein or so claimed
with the phrase "comprising" are expressly or inherently unambiguously
described, enabled and
supported herein for the phrases "consisting essentially of' and "consisting
of."
1006281 While aspects of the invention have been described with reference to
at least one exemplary
embodiment, it is to be clearly understood by those skilled in the art that
the invention is not limited
thereto. Rather, the scope of the invention is to be interpreted only in
conjunction with the appended
claims and it is made clear, here, that the inventor(s) believe that the
claimed subject matter is the
invention.
REFERENCES
1006291 The references disclosed in the specification and Examples, including
but not limited to
patents and patent applications, and international patent applications are all
incorporated herein in
their entirety by reference.
1006301 All patents, patent publications, and other publications referenced
and identified in the
present specification are individually and expressly incorporated herein by
reference in their entirety
for the purpose of describing and disclosing, for example, the compositions
and methodologies
described in such publications that might be used in connection with the
present invention. These
publications am provided solely for their disclosure prior to the filing date
of the present application.
Nothing in this regard should be construed as an admission that the inventors
are not entitled to
antedate such disclosure by virtue of prior invention or for any other reason.
All statements as to the
date or representation as to the contents of these documents is based on the
information available to
the applicants and does not constitute any admission as to the correctness of
the dates or contents of
these documents.
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